• 1 Overview
  • 2 Over-representation analysis (ORA)
    • 2.1 import results of differential expression analysis
    • 2.2 manually calculate enrichment of Reactome geneset “Hemostasis” in Y640F.vs.dsRed
      • 2.2.1 download and import Reactome genesets
      • 2.2.2 convert to dataframe and merge with results from differential expression analysis
    • 2.3 extract unique genes from Reactome, und use to subset result from diff.exp.analysis
      • 2.3.1 Add column indicating which genes are in Genset “Hemostasis” in Diff.Exp.Table
      • 2.3.2 Add column indicating which genes sig. diff. regulated (padj & log2FC cutoff)
      • 2.3.3 Check how the overlap between sig. genes and reactome homeostasis genset is
      • 2.3.4 Now use Fisher Exact test to test for sig. enrichment of Homeostasis genes in sig. reg. genes
      • 2.3.5 Manually calculate Odds Ratio
    • 2.4 Run over-representation analysis over different Genset collections using Bioconductor Tool Cluster Profiler
      • 2.4.1 clusterProfiler needs Entrezid, or Ensemblid -> convert symbols to Entrezid
      • 2.4.2 Make vector of EntrezId from sig. genes, and from all genes (== universe)
      • 2.4.3 run ClusterProfiler for ORA of Reactome genesets
      • 2.4.4 extract all pathways
      • 2.4.5 extract sig. pathways
      • 2.4.6 dataframe: only get results with sig padj < 0.05
      • 2.4.7 show sig. genes in Reactome Pathway illustration
      • 2.4.8 use KEGG database to get more similar pathways as in paper
      • 2.4.9 visualize results with different plotting methods provided by ClusterProfiler
      • 2.4.10 use Database for which there are no special functions in ClusterProfiler / or if you need to use self made genesets
      • 2.4.11 clusterProfile provides function to compare pathway enrichments from multiple sig. gene lists
  • 3 perform Functional Class scoring using GSEA implementation from Cluster Profiler
    • 3.0.1 GSEA using gene sets from KEGG pathways
    • 3.0.2 perform GSEA using the hallmark gene set
    • 3.0.3 Most plot methods also work on GSEA output
  • 4 Pathway topology analysis using SPIA
    • 4.1 Run SPIA
    • 4.2 Genemania
      • 4.2.1 explore relationship of our sig. genes to each other, or to possible interactors
    • 4.3 STRING
      • 4.3.1 We use sig. up-regulated genes to perform ORA analysis
      • 4.3.2 write out named, ranked vector used before for GSEA as 2 column dataframe, to use it for string GSEA
  • 5 homework
    • 5.1 perform analysis with STAT3-wt vs. dsRed; compare results to results obtained already in this skript with Y640F vs. dsRed
    • 5.2 perform ORAs, GSEA, SPIA with genes up-reg. by y640f but not/down-reg. by stat3-wt

1 Overview

In this script, we will perform functional analysis of the identified, differential genes.

In the first part, we will do over-representation analysis (ORA).

In the second part, we will do functional class scoring using gene set enrichement analysis (GSEA).

In the third part we will use SPIA, a tool for Pathway topology.

We will then also shortly try a tool that performs network analysis, namely STRING. String is a Webservice.

Figures generated in this session will be part of the poster. As in the previous section, I would like you to save the plots, and put them in a presentation, together with a short figure legends, and some words describing the figure. # define path to input data here, the input data for this skript is saved

inpDir <- "/proj/courses/transcriptomics/SS23/hw/011_clusterProfiler/inputData"

2 Over-representation analysis (ORA)

2.1 import results of differential expression analysis

l_df.res.all <- readRDS(paste(inpDir, "l_df.res.all.rds", sep = "/"))
names(l_df.res.all)
##  [1] "s.vs.c"                  "y.vs.c"                 
##  [3] "s.vs.c.noNa"             "y.vs.c.noNa"            
##  [5] "s.vs.c.noNa.sigPadjUp"   "y.vs.c.noNa.sigPadjUp"  
##  [7] "s.vs.c.noNa.sigPadjDo"   "y.vs.c.noNa.sigPadjDo"  
##  [9] "s.vs.c.noNa.sigPadjFcUp" "y.vs.c.noNa.sigPadjFcUp"
## [11] "s.vs.c.noNa.sigPadjFcDo" "y.vs.c.noNa.sigPadjFcDo"

only y.vs.c.noNa and s.vs.c.noNa are needed (we will subset from full result tables)

df_y.vs.c.noNa <- l_df.res.all$y.vs.c.noNa
df_s.vs.c.noNa <- l_df.res.all$s.vs.c.noNa
dim(df_y.vs.c.noNa)
## [1] 18028     9
dim(df_s.vs.c.noNa)
## [1] 17507     9
head(df_s.vs.c.noNa)
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
ENSMUSG00000016494.10Cd348841.095-3.8819540.069789260.000000e+00
ENSMUSG00000018474.19Chd31595.129-5.5317280.106788160.000000e+00
ENSMUSG00000004040.17Stat319857.0602.1464400.043422140.000000e+00
ENSMUSG00000049804.10Armcx42485.031-2.4318350.056790030.000000e+00
ENSMUSG00000069045.12Ddx3y2610.027-3.2711660.068434220.000000e+00
ENSMUSG00000069049.12Eif2s3y1312.908-3.0525860.088462126.016023e-262

this is the number of sig. reg. genes (padj < 0.05 & abs(log2fc) > 1)

nrow(df_s.vs.c.noNa[df_s.vs.c.noNa$padj < 0.05 & 
                                 abs(df_s.vs.c.noNa$log2FoldChange) > 1, ])
## [1] 840
nrow(df_y.vs.c.noNa[df_y.vs.c.noNa$padj < 0.05 & 
                                 abs(df_y.vs.c.noNa$log2FoldChange) > 1, ])
## [1] 947

2.2 manually calculate enrichment of Reactome geneset “Hemostasis” in Y640F.vs.dsRed

2.2.1 download and import Reactome genesets

Reactome geneset can be downloaded from here linkToMsigDb

You need to register and login to MsigDb

Download m2.cp.reactome.v2023.1.Mm.symbols.gmt in current working directory.

Now read in and check object

library(GSEABase)
gmt_r <- GSEABase::getGmt(paste(inpDir, 
                                "m2.cp.reactome.v2023.1.Mm.symbols.gmt", 
                                sep = "/"))
gmt_r # GeneSetCollection object
## GeneSetCollection
##   names: REACTOME_INTERLEUKIN_6_SIGNALING, REACTOME_APOPTOSIS, ..., REACTOME_ION_CHANNEL_TRANSPORT (1251 total)
##   unique identifiers: Stat3, Il6st, ..., Trak1 (9045 total)
##   types in collection:
##     geneIdType: NullIdentifier (1 total)
##     collectionType: NullCollection (1 total)

2.2.2 convert to dataframe and merge with results from differential expression analysis

The next step is to know in which genset each of the genes tested for differential regulation belongs to. Therefore, we generate a table, with two columns, one geneset, and one gene-symbol column.

First convert GeneSetCollection Object into a list.

l_gs <- geneIds(gmt_r)

Then convert it into a data.frame using functions from the tidyverse universe tidyverse

df_gsToGene <- as.data.frame(unnest(enframe(l_gs, 
                                            name = "geneset", 
                                            value = "symbol"), 
                                    cols = "symbol"))
dim(df_gsToGene)
## [1] 70591     2
sum(duplicated(df_gsToGene$symbol))
## [1] 61546
length(unique(df_gsToGene$symbol))
## [1] 9045
head(df_gsToGene, n=4)
ABCDEFGHIJ0123456789
 
 
geneset
<chr>
symbol
<chr>
1REACTOME_INTERLEUKIN_6_SIGNALINGStat3
2REACTOME_INTERLEUKIN_6_SIGNALINGIl6st
3REACTOME_INTERLEUKIN_6_SIGNALINGJak2
4REACTOME_INTERLEUKIN_6_SIGNALINGIl6
  • Many genes are present in multiple pathways, therefore, much more rows than unique genes. Note that number of unique genes in Reactome is far less than number of unique genes tested for differential expression analysis (9045 vs. ~17500).

2.3 extract unique genes from Reactome, und use to subset result from diff.exp.analysis

The genes in the Reactome database are our “universe”, within we try to find an association between the gensets and our differentially expressed genes. Subset results of differentially expressed genes to genes present in Reactome database.

v_gInR <- unique(df_gsToGene$symbol)

Before subsetting, we check if we have duplicated gene symbols in our result tables

sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 5

Show rows with duplicated gene symbols

df_y.vs.c.noNa[duplicated(df_y.vs.c.noNa$symbol) | 
                 duplicated(df_y.vs.c.noNa$symbol, fromLast=T), ]
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
ENSMUSG00000030695.17Aldoa47325.4627640.359194930.045780372.610032e-15
ENSMUSG00000006378.16Gcat180.2027720.454795380.141347395.070906e-04
ENSMUSG00000022684.16Bfar1389.389277-0.178678740.056042611.216667e-03
ENSMUSG00000079737.6Bfar252.347122-0.379909520.133226352.119518e-03
ENSMUSG00000117869.2Snhg41305.5121280.131923780.082223941.013645e-01
ENSMUSG00000107877.34933427D14Rik71.7870380.241737330.204295521.581147e-01
ENSMUSG00000114515.3Aldoa31.7777790.198122680.265008993.019270e-01
ENSMUSG00000020807.164933427D14Rik9.071639-0.082670230.324533276.006234e-01
ENSMUSG00000116378.2Gcat3.7941640.065590340.343890216.058058e-01
ENSMUSG00000117694.2Snhg411.2243250.067586080.311730566.873047e-01

We see that there are some genes with multiple Ensemble Gene Ids.

We simply remove one of the duplicates randomly, to avoid problems later, with subsetting, merging, …

df_y.vs.c.noNa <- df_y.vs.c.noNa[!duplicated(df_y.vs.c.noNa$symbol), ]
df_s.vs.c.noNa <- df_s.vs.c.noNa[!duplicated(df_s.vs.c.noNa$symbol), ]
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 0
sum(duplicated(df_s.vs.c.noNa$symbol))
## [1] 0

Next, we subset the data.

df_y.vs.c.noNa <- df_y.vs.c.noNa[df_y.vs.c.noNa$symbol %in% v_gInR, ]
df_s.vs.c.noNa <- df_s.vs.c.noNa[df_s.vs.c.noNa$symbol %in% v_gInR, ]
dim(df_y.vs.c.noNa)
## [1] 6171    9
dim(df_s.vs.c.noNa)
## [1] 6107    9
length(v_gInR)
## [1] 9045

Not all of the 9045 genes in the Reactome Database were expressed in our data. We end up with ~ 6150 genes, that were expressed, and that are in the Reactome database.

2.3.1 Add column indicating which genes are in Genset “Hemostasis” in Diff.Exp.Table

These symbols belong to HEMOSTASIS pathway

v_gHemo <- unique(df_gsToGene[grep("HEMOSTASIS", df_gsToGene$geneset), ]$symbol)
length(v_gHemo)
## [1] 628

in diff. exp. table, indicate which genes belong to HEMOSTAIS (==1), and which not (==0)

df_y.vs.c.noNa$isHemostasis <- ifelse(df_y.vs.c.noNa$symbol %in% v_gHemo, 1, 0)
table(df_y.vs.c.noNa$isHemostasis, useNA = "always")
## 
##    0    1 <NA> 
## 5768  403    0

We see that from the 628 genes of the Hemostasis genset, only 403 are in the Diff.Exp.Table

do it also with s.vs.c diff.exp.table

df_s.vs.c.noNa$isHemostasis <- ifelse(df_s.vs.c.noNa$symbol %in% v_gHemo, 1, 0)
table(df_s.vs.c.noNa$isHemostasis, useNA = "always")
## 
##    0    1 <NA> 
## 5712  395    0

2.3.2 Add column indicating which genes sig. diff. regulated (padj & log2FC cutoff)

df_y.vs.c.noNa$isSig <- ifelse(df_y.vs.c.noNa$padj < 0.05 & 
                                 abs(df_y.vs.c.noNa$log2FoldChange) > 1, 1, 0)
table(df_y.vs.c.noNa$isSig, useNA = "always")
## 
##    0    1 <NA> 
## 5758  413    0
df_s.vs.c.noNa$isSig <- ifelse(df_s.vs.c.noNa$padj < 0.05 & 
                                 abs(df_s.vs.c.noNa$log2FoldChange) > 1, 1, 0)
table(df_s.vs.c.noNa$isSig, useNA = "always")
## 
##    0    1 <NA> 
## 5743  364    0

Note that because of subsetting, we only test a fraction of originally identified, sig. genes, for enrichtment in the pathways.

2.3.3 Check how the overlap between sig. genes and reactome homeostasis genset is

Do it just for Y640F vs. dsRed.

taby <- table(df_y.vs.c.noNa$isSig, 
             df_y.vs.c.noNa$isHemostasis)
taby_sum <- addmargins(taby, FUN = sum)
## Margins computed over dimensions
## in the following order:
## 1: 
## 2:
taby_sum
##      
##          0    1  sum
##   0   5408  350 5758
##   1    360   53  413
##   sum 5768  403 6171

do it for STAT3

tabs <- table(df_s.vs.c.noNa$isSig, 
             df_s.vs.c.noNa$isHemostasis)
tabs_sum <- addmargins(tabs, FUN = sum)
## Margins computed over dimensions
## in the following order:
## 1: 
## 2:
tabs_sum
##      
##          0    1  sum
##   0   5385  358 5743
##   1    327   37  364
##   sum 5712  395 6107

In the first row we have the not sig. genes, in the second row the sig. genes, and in the third row we have the sum. In the first column we have the genes that are not in the genset, in the second column we have the genes that are in the genset, and in the third column we have the sum.

2.3.4 Now use Fisher Exact test to test for sig. enrichment of Homeostasis genes in sig. reg. genes

Just use Y640F vs. dsRed

fisher.test(taby)
## 
##  Fisher's Exact Test for Count Data
## 
## data:  taby
## p-value = 1.344e-06
## alternative hypothesis: true odds ratio is not equal to 1
## 95 percent confidence interval:
##  1.638457 3.109312
## sample estimates:
## odds ratio 
##   2.274229

We have a sig. enrichment of homeostasis genes in sig. genes. This is shown by the p-value, but you also have to interpret the odds ratio correctly, to judge if there is an enrichment, or a depletion, of homeostasis genes in the sig. genes. do it also for STAT3

fisher.test(tabs)
## 
##  Fisher's Exact Test for Count Data
## 
## data:  tabs
## p-value = 0.00564
## alternative hypothesis: true odds ratio is not equal to 1
## 95 percent confidence interval:
##  1.15771 2.44158
## sample estimates:
## odds ratio 
##   1.701804

2.3.5 Manually calculate Odds Ratio

The odds is the ratio of nr. event occurs / nr. event does not occur.

Odds of homeostasis genes in sig. genes: nr. homeostasis genes in sig. genes / nr. not homoestasis genes in sig. genes.

Odds of homeostasis genes in not sig. genes: nr. homeostasis genes in not sig. genes / nr. not homoestasis genes in not sig. genes.

odds_sigG <- 53/360
odds_notSigG <- 350/5408
odds_sigG
## [1] 0.1472222
odds_notSigG
## [1] 0.06471893

higher proportion of homeostasis genes in sig. genes than in not sig. genes.

Now calculate Odds ratio

odds_ratio <- odds_sigG/odds_notSigG
odds_ratio
## [1] 2.274794

2.4 Run over-representation analysis over different Genset collections using Bioconductor Tool Cluster Profiler

2.4.1 clusterProfiler needs Entrezid, or Ensemblid -> convert symbols to Entrezid

Use df_y.vs.c.noNa already subsetted to correct universe, to see if we can reproduce our finding.

idy <- bitr(df_y.vs.c.noNa$symbol, 
            fromType = "SYMBOL", 
            toType = c("ENTREZID"), 
            OrgDb = "org.Mm.eg.db")
dim(df_y.vs.c.noNa)
## [1] 6171   11
dim(idy)
## [1] 6153    2
head(idy)
ABCDEFGHIJ0123456789
 
 
SYMBOL
<chr>
ENTREZID
<chr>
1Chd3216848
2Pgrmc153328
3Stat320848
4Itga9104099
5Sgpl120397
6Muc1317063

Some symbols did not match an Entrezid, and were dropped.

Check if NA or duplicated Values.

for(i in seq_along(idy)){
  print(sum(duplicated(idy[, i])))
  print(sum(is.na(idy[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0

No.

Use df_s.vs.c.noNa already subsetted to correct universe, to see if we can reproduce our finding.

ids <- bitr(df_s.vs.c.noNa$symbol, 
            fromType = "SYMBOL", 
            toType = c("ENTREZID"), 
            OrgDb = "org.Mm.eg.db")
dim(df_s.vs.c.noNa)
## [1] 6107   11
dim(ids)
## [1] 6090    2
head(ids)
ABCDEFGHIJ0123456789
 
 
SYMBOL
<chr>
ENTREZID
<chr>
1Cd3412490
2Chd3216848
3Stat320848
4Far2330450
5Mid1ip168041
6Uty22290
for(i in seq_along(ids)){
  print(sum(duplicated(ids[, i])))
  print(sum(is.na(ids[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
### Merge diff.Exp.Table with idy


```r
df_y.vs.c.noNa.m <- merge(x=df_y.vs.c.noNa, 
                          y=ids, 
                          by.x="symbol", 
                          by.y="SYMBOL", 
                          all=F) # only keep symbols present in both tables
dim(df_y.vs.c.noNa.m)
## [1] 6090   12
sum(is.na(df_y.vs.c.noNa.m$ENTREZID))
## [1] 0
sum(duplicated(df_y.vs.c.noNa.m$ENTREZID))
## [1] 0
head(df_y.vs.c.noNa.m)
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
stat
<dbl>
11600012H06Rik475.9103540.060878780.084995650.4618636860.6303114530.7357815
22310033P09Rik689.9220210.090161970.082404950.2615876860.4216860261.1226464
32610008E11Rik285.933198-0.428192370.147514240.0015694040.004987662-3.1615342
42810021J22Rik23.633663-0.310455580.302983970.1340997490.250653216-1.4981290
59130019O22Rik4.3867870.065913550.339816160.6248362230.7684541000.4890077
6A430033K04Rik376.791930-0.013563530.093525640.8810511950.933101263-0.1496368
#do it for stat3
df_s.vs.c.noNa.m <- merge(x=df_s.vs.c.noNa, 
                          y=ids, 
                          by.x="symbol", 
                          by.y="SYMBOL", 
                          all=F) # only keep symbols present in both tables
dim(df_s.vs.c.noNa.m)
## [1] 6090   12
sum(is.na(df_s.vs.c.noNa.m$ENTREZID))
## [1] 0
sum(duplicated(df_s.vs.c.noNa.m$ENTREZID))
## [1] 0
head(df_s.vs.c.noNa.m)
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
11600012H06Rik475.9103540.2162667280.085141828.381174e-032.659101e-02
22310033P09Rik689.9220210.1278209930.082239031.081567e-012.309348e-01
32610008E11Rik285.933198-0.6939562780.152696559.517084e-076.601252e-06
42810021J22Rik23.633663-0.7500124230.394657385.723083e-031.914291e-02
59130019O22Rik4.386787-0.0090219250.305909119.313940e-019.648470e-01
6A430033K04Rik376.791930-0.2344168060.096496091.071650e-023.307717e-02

Looks Good, we lost some genes, but have unique Entrezid, without NA values.

2.4.2 Make vector of EntrezId from sig. genes, and from all genes (== universe)

# extract sig. res
df_y.vs.c.noNa.sig <- df_y.vs.c.noNa.m[df_y.vs.c.noNa.m$padj < 0.05 & 
                                         abs(df_y.vs.c.noNa.m$log2FoldChange) > 1,  ]
# dim(df_y.vs.c.noNa.sig)

v_y.vs.c.sig <- as.character(df_y.vs.c.noNa.sig$ENTREZID)
length(v_y.vs.c.sig)
## [1] 413
# do for up reg. Y, extract sig. res

#do it for STAT3
# extract sig. res
df_s.vs.c.noNa.sig <- df_s.vs.c.noNa.m[df_s.vs.c.noNa.m$padj < 0.05 & 
                                         abs(df_s.vs.c.noNa.m$log2FoldChange) > 1,  ]
# dim(df_y.vs.c.noNa.sig)

v_s.vs.c.sig <- as.character(df_s.vs.c.noNa.sig$ENTREZID)
length(v_s.vs.c.sig)
## [1] 363
# Create background dataset for hypergeometric testing using all genes tested for significance in the results
v_y.vs.c.all <- as.character(df_y.vs.c.noNa.m$ENTREZID)
length(v_y.vs.c.all)
## [1] 6090
#for STAT3
v_s.vs.c.all <- as.character(df_s.vs.c.noNa.m$ENTREZID)
length(v_s.vs.c.all)
## [1] 6090

2.4.3 run ClusterProfiler for ORA of Reactome genesets

r_res <- enrichPathway(gene=v_y.vs.c.sig, 
                   pvalueCutoff = 0.05, 
                   readable=TRUE, # convert EntrezId to Gene symbols in result table
                   organism = "mouse", 
                   universe = v_y.vs.c.all)
class(r_res) # enrichResult
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
#for Stat 3
r_res_s <- enrichPathway(gene=v_s.vs.c.sig, 
                   pvalueCutoff = 0.05, 
                   readable=TRUE, # convert EntrezId to Gene symbols in result table
                   organism = "mouse", 
                   universe = v_s.vs.c.all)
class(r_res_s) # enrichResult
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"

2.4.4 extract all pathways

df_res <- r_res@result
dim(df_res)
## [1] 603   9
#stat3
df_res_s <- r_res_s@result
dim(df_res_s)
## [1] 616   9

2.4.5 extract sig. pathways

dim(df_res[df_res$p.adjust < 0.05, ])
## [1] 13  9
#stat3
dim(df_res_s[df_res_s$p.adjust < 0.05, ])
## [1] 20  9

2.4.6 dataframe: only get results with sig padj < 0.05

df_res <- data.frame(r_res) # dataframe: only get results with sig padj < 0.05
dim(df_res)
## [1] 13  9
#STAT3
df_res_s <- data.frame(r_res_s) # dataframe: only get results with sig padj < 0.05
dim(df_res_s)
## [1] 20  9
colnames(df_res)
## [1] "ID"          "Description" "GeneRatio"   "BgRatio"     "pvalue"     
## [6] "p.adjust"    "qvalue"      "geneID"      "Count"
df_res
ABCDEFGHIJ0123456789
 
 
ID
<chr>
Description
<chr>
GeneRatio
<chr>
R-MMU-109582R-MMU-109582Hemostasis50/389
R-MMU-76002R-MMU-76002Platelet activation, signaling and aggregation30/389
R-MMU-114608R-MMU-114608Platelet degranulation18/389
R-MMU-76005R-MMU-76005Response to elevated platelet cytosolic Ca2+18/389
R-MMU-500792R-MMU-500792GPCR ligand binding21/389
R-MMU-372790R-MMU-372790Signaling by GPCR35/389
R-MMU-6803157R-MMU-6803157Antimicrobial peptides7/389
R-MMU-388396R-MMU-388396GPCR downstream signalling33/389
R-MMU-3000178R-MMU-3000178ECM proteoglycans6/389
R-MMU-375276R-MMU-375276Peptide ligand-binding receptors12/389
#STAT3
colnames(df_res_s)
## [1] "ID"          "Description" "GeneRatio"   "BgRatio"     "pvalue"     
## [6] "p.adjust"    "qvalue"      "geneID"      "Count"
df_res_s
ABCDEFGHIJ0123456789
 
 
ID
<chr>
R-MMU-500792R-MMU-500792
R-MMU-1296071R-MMU-1296071
R-MMU-372790R-MMU-372790
R-MMU-198933R-MMU-198933
R-MMU-388396R-MMU-388396
R-MMU-112316R-MMU-112316
R-MMU-1266738R-MMU-1266738
R-MMU-977068R-MMU-977068
R-MMU-373080R-MMU-373080
R-MMU-373076R-MMU-373076

ClusterProfiler does not give you an odds ratio, to judge if geneset is enriched, or depleted. But you can calculate it by deviding GeneRation / BgRatio.

map_dbl(df_res$GeneRatio, ~ eval(parse(text=.x)))/map_dbl(df_res$BgRatio, ~ eval(parse(text=.x)))
##  [1] 2.119548 2.494002 3.409521 3.284783 2.618702 2.006668 6.161651 2.007367
##  [9] 6.906466 3.388078 2.602437 2.992802 2.418426
#STAT3
map_dbl(df_res_s$GeneRatio, ~ eval(parse(text=.x)))/map_dbl(df_res_s$BgRatio, ~ eval(parse(text=.x)))
##  [1] 3.243290 6.508274 2.334002 4.489381 2.201172 2.664805 2.076731 7.691596
##  [9] 5.150025 2.758942 6.508274 2.563865 2.784553 2.079739 2.079739 2.603309
## [17] 2.682679 4.230378 2.354297 1.677770

These were our results when we calculated it manually:

    
         0    1  sum
  0   5408  350 5758
  1    360   53  413
  sum 5768  403 6171
Fisher's Exact Test for Count Data

data:  tab
p-value = 1e-06
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
 1.6 3.1
sample estimates:
odds ratio 
       2.3 

The results are a bit different. For manual calculation, we used gene symbols, ClusterProfiler used EntrezIds. Furthermore, we possibly used different versions of Reactome Database.

2.4.7 show sig. genes in Reactome Pathway illustration

To show fold change in Reactome Pathway illustration, a vector of log2fc of sig. genes, with genes named by Entrezid, is needed.

v_nam <- df_y.vs.c.noNa.sig$log2FoldChange
names(v_nam) <- df_y.vs.c.noNa.sig$ENTREZID
v_nam[1:4]
##     56325     76491     66082     11363 
##  1.792428  1.067964 -1.808291 -5.136837
#STAT3
v_nam_s <- df_s.vs.c.noNa.sig$log2FoldChange
names(v_nam_s) <- df_s.vs.c.noNa.sig$ENTREZID
v_nam_s[1:4]
##     56325     66082     11363    264895 
##  1.862131 -3.692707 -3.571212 -2.117135

Now plot Reactome pathway Hemostasis.

viewPathway("Hemostasis", 
            readable = T, 
            foldChange = v_nam, 
            organism = "mouse")

#STAT3
viewPathway("Hemostasis", 
            readable = T, 
            foldChange = v_nam_s, 
            organism = "mouse")

Genes up or down-regulated are indicated. Brown circles indicate genes not within sig. genes. Gray lines indicates known interactions.

Here is how the pathway looks in Reactome pathway browser pathwayBrowser.

Note that some of our enriched Pathways are in the hirarchy below Hemostasis. So probably there are similar genes in some of the pathways. Plot heatmap.

heatplot(r_res, foldChange=v_nam, showCategory=12) + 
  theme(axis.text.x = element_text(size = 4))

#STAT3
heatplot(r_res_s, foldChange=v_nam, showCategory=12) + 
  theme(axis.text.x = element_text(size = 4))

also plot classical Reactome pathway, and check how it looks with pathview

viewPathway("Platelet calcium homeostasis", 
            readable = T, 
            foldChange = v_nam, 
            organism = "mouse")

#STAT3
viewPathway("Platelet calcium homeostasis", 
            readable = T, 
            foldChange = v_nam_s, 
            organism = "mouse")

This is how it looks in the Reactome Pathway browser link

2.4.8 use KEGG database to get more similar pathways as in paper

reprocess diff.exp.tables (now they are subsetted to genes in Reactome Pathway)

df_y.vs.c.noNa <- l_df.res.all$y.vs.c.noNa
df_s.vs.c.noNa <- l_df.res.all$s.vs.c.noNa
dim(df_y.vs.c.noNa)
## [1] 18028     9
dim(df_s.vs.c.noNa)
## [1] 17507     9

remove duplicated gene symbols, if there

sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 5
df_y.vs.c.noNa <- df_y.vs.c.noNa[!duplicated(df_y.vs.c.noNa$symbol), ]
sum(duplicated(df_y.vs.c.noNa$symbol))
## [1] 0

cluster profiles needs Enrezid -> convert symbols

# 
idy.y <- bitr(df_y.vs.c.noNa$symbol, 
            fromType = "SYMBOL", 
            toType = c("ENTREZID"), 
            OrgDb = "org.Mm.eg.db")

head(idy.y)
ABCDEFGHIJ0123456789
 
 
SYMBOL
<chr>
ENTREZID
<chr>
1Chd3216848
2Pgrmc153328
3Stat320848
4Itga9104099
5Sgpl120397
6Muc1317063
for(i in seq_along(idy.y)){
  print(sum(duplicated(idy.y[, i])))
  print(sum(is.na(idy.y[, i])))
}
## [1] 1
## [1] 0
## [1] 0
## [1] 0

remove duplicated symbol

idy.y <- idy.y[!duplicated(idy.y$SYMBOL), ]
for(i in seq_along(idy.y)){
  print(sum(duplicated(idy.y[, i])))
  print(sum(is.na(idy.y[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
ids.s <- bitr(df_s.vs.c.noNa$symbol, 
            fromType = "SYMBOL", 
            toType = c("ENTREZID"), 
            OrgDb = "org.Mm.eg.db")

head(ids.s)
ABCDEFGHIJ0123456789
 
 
SYMBOL
<chr>
ENTREZID
<chr>
1Cd3412490
2Chd3216848
3Stat320848
4Armcx4100503043
5Ddx3y26900
6Eif2s3y26908
for(i in seq_along(ids.s)){
  print(sum(duplicated(ids.s[, i])))
  print(sum(is.na(ids.s[, i])))
}
## [1] 1
## [1] 0
## [1] 0
## [1] 0

remove duplicated symbol

ids.s <- ids.s[!duplicated(ids.s$SYMBOL), ]

check if all duplicated values removed

# 
for(i in seq_along(idy.y)){
  print(sum(duplicated(idy.y[, i])))
  print(sum(is.na(idy.y[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
dim(idy.y)
## [1] 16235     2
for(i in seq_along(ids.s)){
  print(sum(duplicated(ids.s[, i])))
  print(sum(is.na(ids.s[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
dim(ids.s)
## [1] 15843     2

merge diff.exp.Table with EntrezToSymbol conversion table

df_y.vs.c.noNa.m <- merge(x=df_y.vs.c.noNa, 
                          y=idy.y, 
                          by.x="symbol", 
                          by.y="SYMBOL", 
                          all=F)
dim(df_y.vs.c.noNa.m)
## [1] 16235    10
head(df_y.vs.c.noNa.m)
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
stat
<dbl>
10610009B22Rik232.1673550.268940480.120568110.017663580.044276852.3725958
20610009E02Rik9.4877150.104649180.319496630.537404340.697051970.6167429
30610009L18Rik18.3547380.220984460.305327000.263942600.424322911.1171210
40610010K14Rik15.129557-0.028234050.285134650.871454740.92802801-0.1618109
50610030E20Rik1081.7249940.028362260.070198580.680845220.807996690.4113100
60610033M10Rik1.6460610.046189910.353452410.621901680.766241350.4931570
df_s.vs.c.noNa.m <- merge(x=df_s.vs.c.noNa, 
                          y=ids.s, 
                          by.x="symbol", 
                          by.y="SYMBOL", 
                          all=F)
dim(df_s.vs.c.noNa.m)
## [1] 15848    10
head(df_s.vs.c.noNa.m)
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
10610009B22Rik232.1673550.1112591020.118130523.118127e-015.053138e-01
20610009E02Rik9.487715-0.0994228540.303032844.788268e-016.688599e-01
30610009L18Rik18.3547380.0465906110.257537147.718249e-018.809140e-01
40610010K14Rik15.1295570.0016062640.265575139.932166e-019.969180e-01
50610030E20Rik1081.7249940.4677297010.070411571.100300e-111.363266e-10
60610038B21Rik40.3268660.1191890810.230409334.776907e-016.677969e-01

check for duplicated and NA after merging

# 
for(i in c("symbol", "ENTREZID")){
  print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
  print(sum(is.na(df_y.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
for(i in c("symbol", "ENTREZID")){
  print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
  print(sum(is.na(df_s.vs.c.noNa.m[, i])))
}
## [1] 5
## [1] 0
## [1] 5
## [1] 0

make unique for gene symbol and entrezid

# 
df_y.vs.c.noNa.m <- df_y.vs.c.noNa.m[!duplicated(df_y.vs.c.noNa.m$ENTREZID) & 
                                       !is.na(df_y.vs.c.noNa.m$ENTREZID), ]

df_s.vs.c.noNa.m <- df_s.vs.c.noNa.m[!duplicated(df_s.vs.c.noNa.m$ENTREZID) & 
                                       !is.na(df_s.vs.c.noNa.m$ENTREZID), ]

evaluate that removing of duplicates and NA values worked

for(i in c("symbol", "ENTREZID")){
  print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
  print(sum(is.na(df_y.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0
for(i in c("symbol", "ENTREZID")){
  print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
  print(sum(is.na(df_s.vs.c.noNa.m[, i])))
}
## [1] 0
## [1] 0
## [1] 0
## [1] 0

extract sig. res, and make character vector with Entrezid as names

# extract sig. res
df_y.vs.c.noNa.sig <- df_y.vs.c.noNa.m[df_y.vs.c.noNa.m$padj < 0.05 & 
                                         abs(df_y.vs.c.noNa.m$log2FoldChange) > 1,  ]
dim(df_y.vs.c.noNa.sig)
## [1] 898  10
v_y.vs.c.sig <- unique(as.character(df_y.vs.c.noNa.sig$ENTREZID))
length(v_y.vs.c.sig)
## [1] 898
sum(duplicated(names(v_y.vs.c.sig)))
## [1] 0
df_s.vs.c.noNa.sig <- df_s.vs.c.noNa.m[df_s.vs.c.noNa.m$padj < 0.05 & 
                                         abs(df_s.vs.c.noNa.m$log2FoldChange) > 1,  ]
dim(df_s.vs.c.noNa.sig)
## [1] 802  10
v_s.vs.c.sig <- as.character(df_s.vs.c.noNa.sig$ENTREZID)
length(v_s.vs.c.sig)
## [1] 802
sum(duplicated(names(v_s.vs.c.sig)))
## [1] 0

Create background dataset for hypergeometric testing using all genes tested for significance in the results

# 
v_y.vs.c.all <- as.character(df_y.vs.c.noNa.m$ENTREZID)
v_s.vs.c.all <- as.character(df_s.vs.c.noNa.m$ENTREZID)
length(v_y.vs.c.all)
## [1] 16235
length(v_s.vs.c.all)
## [1] 15843

create common background for s.vs.c and y.vs.c, to compare enriched Kegg Pathways of these comparisions (s. below)

# 
v_y.vs.c.all <- unique(c(v_s.vs.c.all, v_y.vs.c.all))
length(v_y.vs.c.all)
## [1] 16235

run Kegg (for now only on Y640F vs. dsRed)

kky <- enrichKEGG(gene         = v_y.vs.c.sig,
                 organism     = 'mmu',
                 pvalueCutoff = 0.05, 
                 universe = v_y.vs.c.all)
kky <- setReadable(kky, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(kky)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
#STAT3
kks <- enrichKEGG(gene         = v_s.vs.c.sig,
                 organism     = 'mmu',
                 pvalueCutoff = 0.05, 
                 universe = v_s.vs.c.all)
kks <- setReadable(kks, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(kks)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"

extract result table, and check which pathways are enriched

df_kky <- kky@result
df_kky
ABCDEFGHIJ0123456789
 
 
ID
<chr>
mmu04514mmu04514
mmu04630mmu04630
mmu04080mmu04080
mmu04640mmu04640
mmu04020mmu04020
mmu00982mmu00982
mmu04610mmu04610
mmu00980mmu00980
mmu05320mmu05320
mmu05200mmu05200
#STAT3
df_kks <- kks@result
df_kks
ABCDEFGHIJ0123456789
 
 
ID
<chr>
mmu04640mmu04640
mmu04060mmu04060
mmu04514mmu04514
mmu05204mmu05204
mmu00982mmu00982
mmu00980mmu00980
mmu05320mmu05320
mmu05330mmu05330
mmu05150mmu05150
mmu04061mmu04061

subset to pathways with sig. p.adjust

# dim(data.frame(kk))
# max(data.frame(kk)$p.adjust)
# data.frame(enrichResult) gives you the sig. gs. with p.adjust < 0.05
df_kky.sig <- df_kky[df_kky$p.adjust < 0.05, ]
as.matrix(df_kky.sig$Description)
##       [,1]                                                                       
##  [1,] "Cell adhesion molecules - Mus musculus (house mouse)"                     
##  [2,] "JAK-STAT signaling pathway - Mus musculus (house mouse)"                  
##  [3,] "Neuroactive ligand-receptor interaction - Mus musculus (house mouse)"     
##  [4,] "Hematopoietic cell lineage - Mus musculus (house mouse)"                  
##  [5,] "Calcium signaling pathway - Mus musculus (house mouse)"                   
##  [6,] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"           
##  [7,] "Complement and coagulation cascades - Mus musculus (house mouse)"         
##  [8,] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"
##  [9,] "Autoimmune thyroid disease - Mus musculus (house mouse)"                  
## [10,] "Pathways in cancer - Mus musculus (house mouse)"                          
## [11,] "Allograft rejection - Mus musculus (house mouse)"                         
## [12,] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"      
## [13,] "Graft-versus-host disease - Mus musculus (house mouse)"                   
## [14,] "Transcriptional misregulation in cancer - Mus musculus (house mouse)"     
## [15,] "PI3K-Akt signaling pathway - Mus musculus (house mouse)"                  
## [16,] "Intestinal immune network for IgA production - Mus musculus (house mouse)"
## [17,] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"       
## [18,] "Asthma - Mus musculus (house mouse)"
#STAT3
df_kks.sig <- df_kks[df_kks$p.adjust < 0.05, ]
as.matrix(df_kks.sig$Description)
##       [,1]                                                                                        
##  [1,] "Hematopoietic cell lineage - Mus musculus (house mouse)"                                   
##  [2,] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"                       
##  [3,] "Cell adhesion molecules - Mus musculus (house mouse)"                                      
##  [4,] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"                        
##  [5,] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"                            
##  [6,] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"                 
##  [7,] "Autoimmune thyroid disease - Mus musculus (house mouse)"                                   
##  [8,] "Allograft rejection - Mus musculus (house mouse)"                                          
##  [9,] "Staphylococcus aureus infection - Mus musculus (house mouse)"                              
## [10,] "Viral protein interaction with cytokine and cytokine receptor - Mus musculus (house mouse)"
## [11,] "Phagosome - Mus musculus (house mouse)"                                                    
## [12,] "Leishmaniasis - Mus musculus (house mouse)"                                                
## [13,] "Inflammatory bowel disease - Mus musculus (house mouse)"                                   
## [14,] "Pathways in cancer - Mus musculus (house mouse)"                                           
## [15,] "Graft-versus-host disease - Mus musculus (house mouse)"                                    
## [16,] "Type I diabetes mellitus - Mus musculus (house mouse)"                                     
## [17,] "Viral myocarditis - Mus musculus (house mouse)"                                            
## [18,] "Intestinal immune network for IgA production - Mus musculus (house mouse)"                 
## [19,] "Fluid shear stress and atherosclerosis - Mus musculus (house mouse)"                       
## [20,] "Steroid hormone biosynthesis - Mus musculus (house mouse)"                                 
## [21,] "Asthma - Mus musculus (house mouse)"                                                       
## [22,] "JAK-STAT signaling pathway - Mus musculus (house mouse)"                                   
## [23,] "Complement and coagulation cascades - Mus musculus (house mouse)"                          
## [24,] "Rheumatoid arthritis - Mus musculus (house mouse)"                                         
## [25,] "Antigen processing and presentation - Mus musculus (house mouse)"                          
## [26,] "Tuberculosis - Mus musculus (house mouse)"                                                 
## [27,] "Axon guidance - Mus musculus (house mouse)"                                                
## [28,] "Ascorbate and aldarate metabolism - Mus musculus (house mouse)"                            
## [29,] "Amoebiasis - Mus musculus (house mouse)"                                                   
## [30,] "Th17 cell differentiation - Mus musculus (house mouse)"                                    
## [31,] "Bile secretion - Mus musculus (house mouse)"

2.4.9 visualize results with different plotting methods provided by ClusterProfiler

get the similarity matrix between terms by calculating Jaccard similarity coefficient (used by some plots later for clustering of genesets).

kk2y <- pairwise_termsim(kky)
#STAT3
kk2s <- pairwise_termsim(kks)

In general, plots are ggplots, and can be modified using ggplot functions

Barplot

barplot(kky, showCategory = 10) 

#STAT3
barplot(kks, showCategory = 10) 

dotplot

dotplot(kky, showCategory=12) + 
  ggtitle("dotplot for ORA") + 
  theme(axis.text.y = element_text(size = 7))

#STAT3
dotplot(kks, showCategory=12) + 
  ggtitle("dotplot for ORA") + 
  theme(axis.text.y = element_text(size = 7))

df_kky.sig[df_kky.sig$Description %in% "Allograft rejection - Mus musculus (house mouse)", ]
ABCDEFGHIJ0123456789
 
 
ID
<chr>
Description
<chr>
GeneRatio
<chr>
BgRatio
<chr>
mmu05330mmu05330Allograft rejection - Mus musculus (house mouse)7/37022/5184
#STAT3
df_kks.sig[df_kks.sig$Description %in% "Allograft rejection - Mus musculus (house mouse)", ]
ABCDEFGHIJ0123456789
 
 
ID
<chr>
Description
<chr>
GeneRatio
<chr>
BgRatio
<chr>
mmu05330mmu05330Allograft rejection - Mus musculus (house mouse)8/31021/5125

heatmap

heatplot(kky, foldChange=v_nam, showCategory=12) + 
  theme(axis.text.x = element_text(size = 3.75))

#STAT3
heatplot(kks, foldChange=v_nam_s, showCategory=12) + 
  theme(axis.text.x = element_text(size = 3.75))

treeplot

treeplot(kk2y, hclust_method = "average")

#STAT3
treeplot(kk2s, hclust_method = "average")

cnetplot

cnetplot(kk2y, categorySize = "pvalue", foldChange = v_nam, 
         showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5) 

#STAT3
cnetplot(kk2s, categorySize = "pvalue", foldChange = v_nam_s, 
         showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5)

enrichplot

enrichplot::emapplot(kk2y, 
                     # cex_category=1.5,
                     layout="kk", 
                     showCategory=18, 
                     cex_label_category = 0.5)

#STAT3
enrichplot::emapplot(kk2s, 
                     # cex_category=1.5,
                     layout="kk", 
                     showCategory=18, 
                     cex_label_category = 0.5)

genesets with common genes cluster together, and are connected with edges. Thickness of edges correlates with proportion of overlapping genes.

Enrichplot can be further manipulated in an interactive way using the Cytoscape Plugin Enrichment Map link. Another Possibility to visualises clusters of similar pathways as an enrichment network is aPEAR link.

You can also access a specific pathway in the the Kegg pathway browser

browseKEGG(kky, 'mmu04630')
#STAT3
browseKEGG(kks, 'mmu04630')

or visualize a pathway, including the fold change of sig. genes in the pathway

mmu04630 <- pathview(gene.data  = v_nam,
                     pathway.id = "mmu04630",
                     species    = "mmu",
                     limit      = list(gene=max(abs(v_nam)), cpd=1))
#STAT
mmu04630_s <- pathview(gene.data  = v_nam_s,
                     pathway.id = "mmu04630",
                     species    = "mmu",
                     limit      = list(gene=max(abs(v_nam_s)), cpd=1))
# png is saved in current directory

mmu04630

2.4.10 use Database for which there are no special functions in ClusterProfiler / or if you need to use self made genesets

load hallmark GMT file from MsigDb

file was downloaded from here msigdb.

gmt_h <- GSEABase::getGmt(paste(inpDir, "mh.all.v2023.1.Mm.entrez.gmt", sep = "/"))

gmt_h # GeneSetCollection object
## GeneSetCollection
##   names: HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_HYPOXIA, ..., HALLMARK_PANCREAS_BETA_CELLS (50 total)
##   unique identifiers: 23849, 16449, ..., 18088 (4289 total)
##   types in collection:
##     geneIdType: NullIdentifier (1 total)
##     collectionType: NullCollection (1 total)

convert to list of genesets

l_gs <- geneIds(gmt_h)

convert to data.frame in long format

df_gsToGene <- as.data.frame(unnest(enframe(l_gs, name = "geneset", value = "symbol"), 
                                    cols = "symbol"))
dim(df_gsToGene)
## [1] 7189    2
head(df_gsToGene)
ABCDEFGHIJ0123456789
 
 
geneset
<chr>
symbol
<chr>
1HALLMARK_TNFA_SIGNALING_VIA_NFKB23849
2HALLMARK_TNFA_SIGNALING_VIA_NFKB16449
3HALLMARK_TNFA_SIGNALING_VIA_NFKB19347
4HALLMARK_TNFA_SIGNALING_VIA_NFKB14528
5HALLMARK_TNFA_SIGNALING_VIA_NFKB14595
6HALLMARK_TNFA_SIGNALING_VIA_NFKB15894

nr of genes in Hallmark

(l_g_hm <- length(unique(df_gsToGene$symbol)))
## [1] 4289

nr of genes in diff.exp.table and hallmark

(l_is_hm.deg <- length(intersect(unique(df_gsToGene$symbol), 
                         v_y.vs.c.all)))
## [1] 3195
#Stat3
(l_is_hm.deg_s <- length(intersect(unique(df_gsToGene$symbol), 
                         v_s.vs.c.all)))
## [1] 3169

ClusterProfiler has function “enricher” for performing over-representation analysis of various gensets. As an input, it needs again the vector of sig. genes, IDs as Entrezid, a dataframe with two columns names “geneset” and “symbol”, and some additional, optional parameters. We also define the background genset, defined by the parameter “universe”, taking all genes that were tested.

hy <- enricher(v_y.vs.c.sig, 
              TERM2GENE = df_gsToGene, 
              universe = v_y.vs.c.all)
class(hy)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
data.frame(hy)
ABCDEFGHIJ0123456789
 
 
ID
<chr>
HALLMARK_INTERFERON_GAMMA_RESPONSEHALLMARK_INTERFERON_GAMMA_RESPONSE
HALLMARK_INFLAMMATORY_RESPONSEHALLMARK_INFLAMMATORY_RESPONSE
HALLMARK_ANGIOGENESISHALLMARK_ANGIOGENESIS
HALLMARK_IL2_STAT5_SIGNALINGHALLMARK_IL2_STAT5_SIGNALING
HALLMARK_HYPOXIAHALLMARK_HYPOXIA
HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITIONHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION
HALLMARK_INTERFERON_ALPHA_RESPONSEHALLMARK_INTERFERON_ALPHA_RESPONSE
HALLMARK_TNFA_SIGNALING_VIA_NFKBHALLMARK_TNFA_SIGNALING_VIA_NFKB
HALLMARK_HEME_METABOLISMHALLMARK_HEME_METABOLISM
HALLMARK_KRAS_SIGNALING_UPHALLMARK_KRAS_SIGNALING_UP
#STAT3
hs <- enricher(v_s.vs.c.sig, 
              TERM2GENE = df_gsToGene, 
              universe = v_s.vs.c.all)
class(hs)
## [1] "enrichResult"
## attr(,"package")
## [1] "DOSE"
data.frame(hs)
ABCDEFGHIJ0123456789
 
 
ID
<chr>
HALLMARK_INTERFERON_GAMMA_RESPONSEHALLMARK_INTERFERON_GAMMA_RESPONSE
HALLMARK_KRAS_SIGNALING_UPHALLMARK_KRAS_SIGNALING_UP
HALLMARK_IL6_JAK_STAT3_SIGNALINGHALLMARK_IL6_JAK_STAT3_SIGNALING
HALLMARK_HYPOXIAHALLMARK_HYPOXIA
HALLMARK_INFLAMMATORY_RESPONSEHALLMARK_INFLAMMATORY_RESPONSE
HALLMARK_ESTROGEN_RESPONSE_EARLYHALLMARK_ESTROGEN_RESPONSE_EARLY
HALLMARK_INTERFERON_ALPHA_RESPONSEHALLMARK_INTERFERON_ALPHA_RESPONSE
HALLMARK_IL2_STAT5_SIGNALINGHALLMARK_IL2_STAT5_SIGNALING
  • 12 hallmark genesets with a sig. p.adjust
barplot(hy, categories=12)

#Stat3
barplot(hs, categories=12)

2.4.11 clusterProfile provides function to compare pathway enrichments from multiple sig. gene lists

compare Y640F.vs.dsRed with STAT3-wt.vs.dsRed. Put sig. genelists into a list.

l_sigEntrez <- list(y.vs.c = v_y.vs.c.sig, 
                    s.vs.c = v_s.vs.c.sig)

Run function compareCluster

ck <- compareCluster(geneCluster = l_sigEntrez, 
                     fun="enrichKEGG", 
                     organism="mmu", pvalueCutoff=0.05, pAdjustMethod="BH")
ck <- setReadable(ck, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
class(ck)
## [1] "compareClusterResult"
## attr(,"package")
## [1] "DOSE"

extract sig. results

res_comp <- data.frame(ck)
dim(res_comp)
## [1] 55 10
colnames(res_comp)
##  [1] "Cluster"     "ID"          "Description" "GeneRatio"   "BgRatio"    
##  [6] "pvalue"      "p.adjust"    "qvalue"      "geneID"      "Count"
head(res_comp, n=10)
ABCDEFGHIJ0123456789
 
 
Cluster
<fct>
ID
<chr>
1y.vs.cmmu04630
2y.vs.cmmu04640
3y.vs.cmmu04151
4y.vs.cmmu05202
5y.vs.cmmu04514
6y.vs.cmmu04015
7y.vs.cmmu04014
8y.vs.cmmu04668
9y.vs.cmmu04658
10y.vs.cmmu01521
tail(res_comp, n=10)
ABCDEFGHIJ0123456789
 
 
Cluster
<fct>
ID
<chr>
Description
<chr>
46s.vs.cmmu04612Antigen processing and presentation - Mus musculus (house mouse)
47s.vs.cmmu05202Transcriptional misregulation in cancer - Mus musculus (house mouse)
48s.vs.cmmu05145Toxoplasmosis - Mus musculus (house mouse)
49s.vs.cmmu05166Human T-cell leukemia virus 1 infection - Mus musculus (house mouse)
50s.vs.cmmu04151PI3K-Akt signaling pathway - Mus musculus (house mouse)
51s.vs.cmmu05320Autoimmune thyroid disease - Mus musculus (house mouse)
52s.vs.cmmu05169Epstein-Barr virus infection - Mus musculus (house mouse)
53s.vs.cmmu05332Graft-versus-host disease - Mus musculus (house mouse)
54s.vs.cmmu05163Human cytomegalovirus infection - Mus musculus (house mouse)
55s.vs.cmmu04014Ras signaling pathway - Mus musculus (house mouse)
unique(res_comp$Description)
##  [1] "JAK-STAT signaling pathway - Mus musculus (house mouse)"                                   
##  [2] "Hematopoietic cell lineage - Mus musculus (house mouse)"                                   
##  [3] "PI3K-Akt signaling pathway - Mus musculus (house mouse)"                                   
##  [4] "Transcriptional misregulation in cancer - Mus musculus (house mouse)"                      
##  [5] "Cell adhesion molecules - Mus musculus (house mouse)"                                      
##  [6] "Rap1 signaling pathway - Mus musculus (house mouse)"                                       
##  [7] "Ras signaling pathway - Mus musculus (house mouse)"                                        
##  [8] "TNF signaling pathway - Mus musculus (house mouse)"                                        
##  [9] "Th1 and Th2 cell differentiation - Mus musculus (house mouse)"                             
## [10] "EGFR tyrosine kinase inhibitor resistance - Mus musculus (house mouse)"                    
## [11] "Calcium signaling pathway - Mus musculus (house mouse)"                                    
## [12] "Intestinal immune network for IgA production - Mus musculus (house mouse)"                 
## [13] "Toxoplasmosis - Mus musculus (house mouse)"                                                
## [14] "Acute myeloid leukemia - Mus musculus (house mouse)"                                       
## [15] "Melanoma - Mus musculus (house mouse)"                                                     
## [16] "Epstein-Barr virus infection - Mus musculus (house mouse)"                                 
## [17] "Hepatitis C - Mus musculus (house mouse)"                                                  
## [18] "Asthma - Mus musculus (house mouse)"                                                       
## [19] "Inflammatory bowel disease - Mus musculus (house mouse)"                                   
## [20] "Focal adhesion - Mus musculus (house mouse)"                                               
## [21] "Leishmaniasis - Mus musculus (house mouse)"                                                
## [22] "Cytokine-cytokine receptor interaction - Mus musculus (house mouse)"                       
## [23] "Phagosome - Mus musculus (house mouse)"                                                    
## [24] "Viral protein interaction with cytokine and cytokine receptor - Mus musculus (house mouse)"
## [25] "Tuberculosis - Mus musculus (house mouse)"                                                 
## [26] "Fluid shear stress and atherosclerosis - Mus musculus (house mouse)"                       
## [27] "Th17 cell differentiation - Mus musculus (house mouse)"                                    
## [28] "Rheumatoid arthritis - Mus musculus (house mouse)"                                         
## [29] "Axon guidance - Mus musculus (house mouse)"                                                
## [30] "Allograft rejection - Mus musculus (house mouse)"                                          
## [31] "MAPK signaling pathway - Mus musculus (house mouse)"                                       
## [32] "Kaposi sarcoma-associated herpesvirus infection - Mus musculus (house mouse)"              
## [33] "Chemical carcinogenesis - DNA adducts - Mus musculus (house mouse)"                        
## [34] "Drug metabolism - cytochrome P450 - Mus musculus (house mouse)"                            
## [35] "Viral myocarditis - Mus musculus (house mouse)"                                            
## [36] "Metabolism of xenobiotics by cytochrome P450 - Mus musculus (house mouse)"                 
## [37] "Amoebiasis - Mus musculus (house mouse)"                                                   
## [38] "Antigen processing and presentation - Mus musculus (house mouse)"                          
## [39] "Human T-cell leukemia virus 1 infection - Mus musculus (house mouse)"                      
## [40] "Autoimmune thyroid disease - Mus musculus (house mouse)"                                   
## [41] "Graft-versus-host disease - Mus musculus (house mouse)"                                    
## [42] "Human cytomegalovirus infection - Mus musculus (house mouse)"

get distance matrix for plots

ck2 <- pairwise_termsim(ck)

dotplot

dotplot(ck, showCategory = 40, font.size = 8)

dotplot

dotplot(ck, showCategory=12) + 
  ggtitle("dotplot for ORA") + 
  theme(axis.text.y = element_text(size = 7))

heatmap

heatplot(ck)
## Error in (function (classes, fdef, mtable) : unable to find an inherited method for function 'heatplot' for signature '"compareClusterResult"'

treeplot

treeplot(ck2, hclust_method = "average")

cnetplot

cnetplot(ck2, categorySize = "pvalue",  
         showCategory=5, cex_label_gene=0.5, cex_label_category = 0.5) 

you see common and specific enriched pathways; you see, whether in common pathways, common or specific genes are enriched.

enrichplot

enrichplot::emapplot(ck2, cex_category=1.5,layout="kk", 
                     showCategory=20)

3 perform Functional Class scoring using GSEA implementation from Cluster Profiler

We need to create a ranked list from our diff. exp. analysis. We rank the list based on the -log10(p-value). In addition, we indicate the direction of regulation, by multiplying the minus log10 pvalue with the sign of the fold change.

We use already above prepared dataframe, with unique Entrezid, and no NA values. We check for duplicated and NA values, and 0 values (will be inf when log transformed) in pvalue column. We also check log2FC (there, just NA values would be a problem).

for(i in c("pvalue", "log2FoldChange")){
  print(sum(duplicated(df_y.vs.c.noNa.m[, i])))
  print(sum(is.na(df_y.vs.c.noNa.m[, i])))
  print(sum(df_y.vs.c.noNa.m[, i] == 0))
}
## [1] 2
## [1] 0
## [1] 2
## [1] 1
## [1] 0
## [1] 0
#STAT3
for(i in c("pvalue", "log2FoldChange")){
  print(sum(duplicated(df_s.vs.c.noNa.m[, i])))
  print(sum(is.na(df_s.vs.c.noNa.m[, i])))
  print(sum(df_s.vs.c.noNa.m[, i] == 0))
}
## [1] 4
## [1] 0
## [1] 5
## [1] 0
## [1] 0
## [1] 0

There are 2 duplicated p-values (we dont mind about duplicated log2FC). There are 2 values with p=0. Examine values.

df_y.vs.c.noNa.m[duplicated(df_y.vs.c.noNa.m$pvalue) | duplicated(df_y.vs.c.noNa.m$pvalue, 
                                                                  fromLast = T), ]
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
stat
<dbl>
1840C1qb1.4738390.10239970.371165990.26598390.42688131.112359
2457Chd31595.129092-3.23791150.075346390.00000000.0000000-43.033316
3442Dpep21.4738390.10239970.371165990.26598390.42688131.112359
11154Pgrmc11244.715326-3.71294840.095963560.00000000.0000000-38.778987
#STAT3
df_s.vs.c.noNa.m[duplicated(df_s.vs.c.noNa.m$pvalue) | duplicated(df_s.vs.c.noNa.m$pvalue, 
                                                                  fromLast = T), ]
ABCDEFGHIJ0123456789
 
 
symbol
<chr>
baseMean
<dbl>
log2FoldChange
<dbl>
lfcSE
<dbl>
pvalue
<dbl>
padj
<dbl>
stat
<dbl>
lfcSE_not_shrunk
<dbl>
1241Armcx42485.031-2.4318350.0567900300-42.875830.05677956
2140Cd348841.095-3.8819540.0697892600-55.679070.06976226
2400Chd31595.129-5.5317280.1067881600-51.812340.10678829
3121Ddx3y2610.027-3.2711660.0684342200-47.847610.06842538
13584Stat319857.0602.1464400.043422140049.493580.04340101

We have to add a value, to avoid that 0 becomes Inf after log transformation.

df_y.vs.c.noNa.m$pvalue[which(df_y.vs.c.noNa.m$pvalue == 0)[1]] <- 2.225074e-308
df_y.vs.c.noNa.m$pvalue[which(df_y.vs.c.noNa.m$pvalue == 0)] <- 2.225084e-308
which(df_y.vs.c.noNa.m$pvalue == 0)
## integer(0)
#STAT3
df_s.vs.c.noNa.m$pvalue[which(df_s.vs.c.noNa.m$pvalue == 0)[1]] <- 2.225074e-308
df_s.vs.c.noNa.m$pvalue[which(df_s.vs.c.noNa.m$pvalue == 0)] <- 2.225084e-308
which(df_s.vs.c.noNa.m$pvalue == 0)
## integer(0)

We changed pvalues from 0 to small values.

If you have more p-values == 0, then use “stat” column from DESeq2 output, instead of p-value.

Make ranked gene list.

v_namedRnk <- -log10(df_y.vs.c.noNa.m$pvalue) * sign(df_y.vs.c.noNa.m$log2FoldChange)
#Stat3
v_namedRnk_s <- -log10(df_s.vs.c.noNa.m$pvalue) * sign(df_s.vs.c.noNa.m$log2FoldChange)

name vector by Entrez ID

names(v_namedRnk) <- df_y.vs.c.noNa.m$ENTREZID
#STAT3
names(v_namedRnk_s) <- df_s.vs.c.noNa.m$ENTREZID

sort decreasing

v_namedRnk <- sort(v_namedRnk, decreasing = T)
#STAT
v_namedRnk_s <- sort(v_namedRnk_s, decreasing = T)

3.0.1 GSEA using gene sets from KEGG pathways

gseaKEGG <- gseKEGG(geneList = v_namedRnk, # ordered named vector
                    organism = "mmu", 
                    minGSSize = 120, # minimum gene set size (# genes in set) - change to test more sets or recover sets with fewer # genes
                    pvalueCutoff = 0.05, # padj cutoff value
                    verbose = FALSE)

## Extract the GSEA results
gseaKEGG_results <- gseaKEGG@result
df_gseaKsig <- gseaKEGG_results[gseaKEGG_results$p.adjust < 0.05, ]
dim(df_gseaKsig)
## [1]  1 11
df_gseaKsig
ABCDEFGHIJ0123456789
 
 
ID
<chr>
Description
<chr>
setSize
<int>
mmu05171mmu05171Coronavirus disease - COVID-19 - Mus musculus (house mouse)180
#STAT3
gseaKEGG_s <- gseKEGG(geneList = v_namedRnk_s, # ordered named vector
                    organism = "mmu", 
                    minGSSize = 120, # minimum gene set size (# genes in set) - change to test more sets or recover sets with fewer # genes
                    pvalueCutoff = 0.05, # padj cutoff value
                    verbose = FALSE)

## Extract the GSEA results
gseaKEGG_results_s <- gseaKEGG_s@result
df_gseaKsig_s <- gseaKEGG_results_s[gseaKEGG_results_s$p.adjust < 0.05, ]
dim(df_gseaKsig_s)
## [1] 0 8
df_gseaKsig_s
ABCDEFGHIJ0123456789
ID
<chr>
Description
<chr>
setSize
<int>
enrichmentScore
<dbl>
NES
<dbl>
pvalue
<dbl>
p.adjust
<dbl>
qvalue
<dbl>

Only one, not very useful, gensets significant.

Write GSEA results to file

write.csv(gseaKEGG_results, "gsea.y.vs.c.kegg.csv", quote=F)

3.0.2 perform GSEA using the hallmark gene set

gseaHm <- GSEA(geneList = v_namedRnk, 
               TERM2GENE = df_gsToGene)
gseaHm <- setReadable(gseaHm, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
df_gsea.h.y.c <- data.frame(gseaHm)
dim(df_gsea.h.y.c)
## [1]  3 11
df_gsea.h.y.c
ABCDEFGHIJ0123456789
 
 
ID
<chr>
HALLMARK_INTERFERON_ALPHA_RESPONSEHALLMARK_INTERFERON_ALPHA_RESPONSE
HALLMARK_TNFA_SIGNALING_VIA_NFKBHALLMARK_TNFA_SIGNALING_VIA_NFKB
HALLMARK_INTERFERON_GAMMA_RESPONSEHALLMARK_INTERFERON_GAMMA_RESPONSE
#STAT3
gseaHm_s <- GSEA(geneList = v_namedRnk_s, 
               TERM2GENE = df_gsToGene)
gseaHm_s <- setReadable(gseaHm_s, OrgDb = org.Mm.eg.db, keyType="ENTREZID")
df_gsea.h.y.c_s <- data.frame(gseaHm)
dim(df_gsea.h.y.c_s)
## [1]  3 11
df_gsea.h.y.c_s
ABCDEFGHIJ0123456789
 
 
ID
<chr>
HALLMARK_INTERFERON_ALPHA_RESPONSEHALLMARK_INTERFERON_ALPHA_RESPONSE
HALLMARK_TNFA_SIGNALING_VIA_NFKBHALLMARK_TNFA_SIGNALING_VIA_NFKB
HALLMARK_INTERFERON_GAMMA_RESPONSEHALLMARK_INTERFERON_GAMMA_RESPONSE

3 gensets significant

3.0.3 Most plot methods also work on GSEA output

gseaHm2 <- pairwise_termsim(gseaHm)
#stat3
gseaHm2_s <- pairwise_termsim(gseaHm_s)

Barplot

barplot(gseaHm, showCategory = 2) 
## Error in barplot.default(gseaHm, showCategory = 2): 'height' must be a vector or a matrix
#stat3
barplot(gseaHm_s, showCategory = 2) 
## Error in barplot.default(gseaHm_s, showCategory = 2): 'height' must be a vector or a matrix

dotplot

dotplot(gseaHm, showCategory=3) + 
  ggtitle("dotplot for ORA") + 
  theme(axis.text.y = element_text(size = 7))

#Stat3
dotplot(gseaHm_s, showCategory=3) + 
  ggtitle("dotplot for ORA") + 
  theme(axis.text.y = element_text(size = 7))

heatmap

heatplot(gseaHm, foldChange=v_nam, showCategory=3) + 
  theme(axis.text.x = element_text(size = 3.75))

#stat3
heatplot(gseaHm_s, foldChange=v_nam, showCategory=3) + 
  theme(axis.text.x = element_text(size = 3.75))

treeplot

treeplot(gseaHm2, hclust_method = "average")
## Error in stats::cutree(hc, nCluster): elements of 'k' must be between 1 and 3

cnetplot

cnetplot(gseaHm2, categorySize = "pvalue", foldChange = v_nam, 
         showCategory=3, cex_label_gene=0.5, cex_label_category = 0.5) 

#STAT3
cnetplot(gseaHm2_s, categorySize = "pvalue", foldChange = v_nam_s, 
         showCategory=3, cex_label_gene=0.5, cex_label_category = 0.5) 

In addition, you can make a gseaplot, showing the rank of the genes of the genset in the ranked gene list from the diff.exp.analysis.

gseaplot(gseaHm, geneSetID = 'HALLMARK_INTERFERON_ALPHA_RESPONSE')

4 Pathway topology analysis using SPIA

The above methods don’t take into account how genes interact with each other (e.g. activation, inhibition, phosphorylation, direct/indirect interaction, ubiquitination, etc). Pathway topology methods this extra information in computing pathway-level statistics.

We will use a tool called SPIA. Here are some links to nice description of this tool: link1, link2, link3

as an input, you need a vector with sig. genes, named with entrez id

v_nam.entrez <- df_y.vs.c.noNa.sig$log2FoldChange
names(v_nam.entrez) <- df_y.vs.c.noNa.sig$ENTREZID

remove na and duplicated values

v_nam.entrez <- v_nam.entrez[!is.na(names(v_nam.entrez))] 
v_nam.entrez <- v_nam.entrez[!duplicated(names(v_nam.entrez))]

as a background we use all genes tested in diff. exp. an

background_genes <- df_y.vs.c.noNa.m$ENTREZID
background_genes <- background_genes[!is.na(background_genes)]
background_genes <- background_genes[!duplicated(background_genes)]

4.1 Run SPIA

SPIA uses KEGG Pathways Dont run it - it takes very long on the server!!!!

# spia_result <- spia(de=v_nam.entrez, 
#                     all=background_genes, 
#                     organism="mmu", 
#                     plots = TRUE)

If plot is TRUE in the last command, a pdf file with Perturbation plots (SPIAPerturbationPlots.pdf) will be saved in the current working directory.

Load results from inpDir

# saveRDS(spia_result, paste(inpDir, "spia_results.rds", sep = "/"))
spia_result <- readRDS(paste(inpDir, "spia_results.rds", sep = "/"))
head(spia_result, n=20)
ABCDEFGHIJ0123456789
 
 
Name
<chr>
ID
<chr>
pSize
<int>
NDE
<int>
pNDE
<dbl>
tA
<dbl>
1Cytokine-cytokine receptor interaction04060125277.634504e-109.874791
2Jak-STAT signaling pathway0463095201.879019e-07-4.073919
3Neuroactive ligand-receptor interaction0408071172.196410e-070.000000
4Transcriptional misregulation in cancer05202133191.283151e-04-2.660223
5Influenza A05164126175.634642e-0421.704201
6Asthma053101354.509771e-040.000000
7Autoimmune thyroid disease053202076.298438e-050.000000
8Leukocyte transendothelial migration046708293.665249e-0246.829991
9Allograft rejection053302178.999028e-050.000000
10Graft-versus-host disease053322271.257236e-040.000000

SPIA outputs a table showing significantly dysregulated pathways based on over-representation and signaling perturbations accumulation. The table shows the following information:

  • pSize: the number of genes on the pathway
  • NDE: the number of DE genes per pathway
  • tA: the observed total perturbation accumulation in the pathway
  • pNDE: the probability to observe at least NDE genes on the pathway using a hypergeometric model (similar to ORA)
  • pPERT: the probability to observe a total accumulation more extreme than tA only by chance
  • pG: the p-value obtained by combining pNDE and pPERT
  • pGFdr and pGFWER are the False Discovery Rate and respectively Bonferroni adjusted global p-values
  • Status: gives the direction in which the pathway is perturbed (activated or inhibited)
  • KEGGLINK gives a web link to the KEGG website that displays the pathway image with the differentially expressed genes highlighted in red

sig. pathways:

spia_result[spia_result$pGFdr < 0.05, ]
ABCDEFGHIJ0123456789
 
 
Name
<chr>
ID
<chr>
pSize
<int>
NDE
<int>
pNDE
<dbl>
tA
<dbl>
1Cytokine-cytokine receptor interaction04060125277.634504e-109.874791
2Jak-STAT signaling pathway0463095201.879019e-07-4.073919
3Neuroactive ligand-receptor interaction0408071172.196410e-070.000000
4Transcriptional misregulation in cancer05202133191.283151e-04-2.660223
5Influenza A05164126175.634642e-0421.704201
6Asthma053101354.509771e-040.000000
7Autoimmune thyroid disease053202076.298438e-050.000000
8Leukocyte transendothelial migration046708293.665249e-0246.829991
9Allograft rejection053302178.999028e-050.000000
10Graft-versus-host disease053322271.257236e-040.000000

A summary Plot of the results can be obtained using the command below. The genesets above the blue and red curve are significant based on the combined p-value obtained by combining pNDE and pPERT.

plotP(spia_result, threshold=0.05)
## Error in if (sum(oky) > 0) {: missing value where TRUE/FALSE needed

4.2 Genemania

Genemania is a Webtool linkToGenemania hat has stored interactions of different types/from different resources, between genes. We can use it to explore functional relationships between our sig. reg. genes, and possible relationships to other genes. Before you start using Genemania, please read the help linkToHelp or one of these tutorials tutorial1, tutorial2.

4.2.1 explore relationship of our sig. genes to each other, or to possible interactors

Gene symbols can be used as input. We will therefore write out our sig. genes (gene Symbols) ordered by p-value. We then can use the top e.g. 50 genes for the analysis in Genemania.

write.xlsx(df_y.vs.c.noNa.sig[order(df_y.vs.c.noNa.sig$pvalue), ][, "symbol", drop = FALSE], 
           "df_y.vs.c.noNa.sig.symbOnly.xlsx")
# copy to inpDir
file.copy("df_y.vs.c.noNa.sig.symbOnly.xlsx", inpDir)
## [1] FALSE

4.3 STRING

STRING linkToString is a Webtool that has stored interactions of different types/from different resources, between genes. We can use it to explore functional relationships between our sig. reg. genes, and possible relationships to other genes.

These are some helpful resources how to work with STRING: linkToTutorial1, linkToPublication.

In addition to identify networks between provided genes, it can also perform ORA and GSEA analysis.

4.3.1 We use sig. up-regulated genes to perform ORA analysis

write.xlsx(l_df.res.all$y.vs.c.noNa.sigPadjFcUp, 
           file = "y.vs.c.noNa.sigPadjFcUp.xlsx")
# copy to inpDir
file.copy("y.vs.c.noNa.sigPadjFcUp.xlsx", inpDir)
## [1] FALSE

open xlsx file, and copy Gene symbols into “multiple protein” search box. Then press Search (organism is detected automatically). Press Continue to confirm correct ID identification.

Ignore the Large Network warning, and press continue.

In the legend tab, you see the inputted genes, with a short description of their functions.
In the settings tab, you can modify different settings. You can switch the network type, the meaning of network edges, and which interaction sources are considered.

Furthermore, you can set the minimum required interaction score.

At the analysis tab, you see the results of the enrichment analysis. You can color the nodes by the Pathways they are in, or you can select a single gene, and check in which genset it is.

4.3.2 write out named, ranked vector used before for GSEA as 2 column dataframe, to use it for string GSEA

fwrite(data.frame(entrezId = names(v_nam.entrez), 
                  rank = v_nam.entrez), 
       file = "twoColEntrezRankForString.csv")
v_namedRnk
##         20848         20397         17063         74016         12984 
##  2.900643e+02  1.799662e+02  1.787236e+02  1.710772e+02  1.659187e+02 
##        383787        171207         59126         76108         18007 
##  1.604836e+02  1.542307e+02  1.516464e+02  1.492305e+02  1.380007e+02 
##         18715        227937        239217        227620         14687 
##  1.343826e+02  1.331897e+02  1.214323e+02  1.212545e+02  1.198017e+02 
##         18712         18439         20527         12048         20910 
##  1.168415e+02  1.117257e+02  1.111728e+02  1.084804e+02  1.032206e+02 
##         14727        244418         20908         19122        104445 
##  1.024602e+02  1.020278e+02  9.973689e+01  9.589364e+01  9.446703e+01 
##         70599         12983         14664         18286         72349 
##  9.203889e+01  9.063597e+01  8.933688e+01  8.800249e+01  8.746978e+01 
##        228775        170750        231147         14127        170718 
##  8.716182e+01  8.633005e+01  8.629724e+01  8.555330e+01  8.513510e+01 
##        320706         20515         18024         20893         20322 
##  8.487650e+01  8.480825e+01  8.441473e+01  8.412078e+01  8.389796e+01 
##        384009        228875         23872         56454         69601 
##  8.363232e+01  8.249228e+01  8.192964e+01  8.009802e+01  7.959839e+01 
##         71648         59043         16909         83768        227695 
##  7.876267e+01  7.767889e+01  7.742366e+01  7.474339e+01  7.377179e+01 
##         65973         19261        230784        319520         14683 
##  7.375109e+01  7.337548e+01  7.333213e+01  7.214022e+01  7.193435e+01 
##         99237        100678         72630         19177         56325 
##  7.163053e+01  7.118235e+01  7.113865e+01  7.076159e+01  7.069012e+01 
##        108115         70433         13836         66865         14824 
##  6.962518e+01  6.952887e+01  6.870699e+01  6.844477e+01  6.756922e+01 
##         56738         18550         73192         14086        228836 
##  6.735634e+01  6.723339e+01  6.722348e+01  6.628765e+01  6.561700e+01 
##         56791         77975         68713         19248         74411 
##  6.531394e+01  6.524916e+01  6.477626e+01  6.393354e+01  6.385771e+01 
##        668158        240057        269252         12702         20933 
##  6.359649e+01  6.355244e+01  6.354866e+01  6.275835e+01  6.262285e+01 
##         20448         18822         14958        277463         11603 
##  6.243049e+01  6.232984e+01  6.210187e+01  6.121452e+01  6.113915e+01 
##         14594         21961         66405         11938         74153 
##  6.112550e+01  6.065119e+01  5.951585e+01  5.941325e+01  5.933646e+01 
##         20878         21816         18034         93679         68366 
##  5.922877e+01  5.863664e+01  5.862589e+01  5.782023e+01  5.670593e+01 
##        320365         68526         56045        227682         19025 
##  5.657473e+01  5.648528e+01  5.580984e+01  5.569253e+01  5.558972e+01 
##        227737        112405         16905         29812         13198 
##  5.553520e+01  5.541805e+01  5.541038e+01  5.521660e+01  5.451840e+01 
##         67603         83921        228136        225870        227746 
##  5.441967e+01  5.397198e+01  5.397032e+01  5.379111e+01  5.376745e+01 
##         18195     100502874        227619        378702         70354 
##  5.372340e+01  5.369805e+01  5.355699e+01  5.344620e+01  5.327181e+01 
##        241447         15902         12125         57740         54601 
##  5.285805e+01  5.285158e+01  5.271611e+01  5.250716e+01  5.249337e+01 
##         84652         55963         12575        626096         20813 
##  5.238341e+01  5.211437e+01  5.154504e+01  5.070324e+01  5.063116e+01 
##         17354         17768         98878        213990         70296 
##  5.058862e+01  5.028738e+01  5.028670e+01  4.997524e+01  4.993445e+01 
##         51944         66681         28042        246727        320632 
##  4.992109e+01  4.986400e+01  4.974827e+01  4.956374e+01  4.913336e+01 
##         71699        228607        107094         99412         58521 
##  4.908270e+01  4.877679e+01  4.850738e+01  4.847553e+01  4.794502e+01 
##         69737         20932        212111         20017         16976 
##  4.791869e+01  4.775671e+01  4.766194e+01  4.762970e+01  4.720179e+01 
##        140858     100042784         67210        227634         72323 
##  4.712937e+01  4.706588e+01  4.687747e+01  4.686177e+01  4.646729e+01 
##        268739         73139        114715         78306         19182 
##  4.594992e+01  4.588193e+01  4.561796e+01  4.524027e+01  4.509923e+01 
##         27368         76938         66861         15273         13046 
##  4.494078e+01  4.454298e+01  4.439680e+01  4.419575e+01  4.415043e+01 
##        209378        269682         17540        107746         30933 
##  4.389721e+01  4.387392e+01  4.360376e+01  4.359043e+01  4.345748e+01 
##        228545         18591         26360         94067         17355 
##  4.326196e+01  4.321940e+01  4.287676e+01  4.287528e+01  4.280516e+01 
##         19159        106840        327959         16569         74747 
##  4.252378e+01  4.252180e+01  4.248253e+01  4.247400e+01  4.244949e+01 
##         81703         71435         17222        104418         67889 
##  4.240461e+01  4.240444e+01  4.226358e+01  4.223295e+01  4.212602e+01 
##         52838         72061         66355         70466        231834 
##  4.208410e+01  4.173450e+01  4.160048e+01  4.150721e+01  4.125282e+01 
##         20744         20239        228012         12236         22722 
##  4.118765e+01  4.113943e+01  4.112687e+01  4.106613e+01  4.100474e+01 
##         13002        228714        277010         71326         96979 
##  4.051290e+01  4.049739e+01  4.041333e+01  4.037598e+01  4.036062e+01 
##         54004         66691        228140         59015         23962 
##  4.018468e+01  4.014030e+01  4.004122e+01  3.995350e+01  3.978407e+01 
##         66926         56743         16190        226153        228026 
##  3.973994e+01  3.971649e+01  3.952966e+01  3.945559e+01  3.943635e+01 
##         18750         13481         18010         72401         76267 
##  3.943057e+01  3.939796e+01  3.938567e+01  3.934904e+01  3.928161e+01 
##         56305         22169         93730         68205        227612 
##  3.906920e+01  3.896166e+01  3.884792e+01  3.857091e+01  3.840760e+01 
##        235380         74192         30059         20402         18438 
##  3.829386e+01  3.822025e+01  3.819042e+01  3.818461e+01  3.797905e+01 
##         66220         52040         15901        246728         59029 
##  3.784638e+01  3.767815e+01  3.765459e+01  3.761177e+01  3.741437e+01 
##        241576         16010        229003         26371         17865 
##  3.733559e+01  3.733105e+01  3.725748e+01  3.715984e+01  3.713662e+01 
##         50755         66616         26569         12578        269261 
##  3.710064e+01  3.700502e+01  3.672083e+01  3.655291e+01  3.654966e+01 
##         12447        227700        269338        213435         84095 
##  3.649882e+01  3.646833e+01  3.644963e+01  3.644500e+01  3.642002e+01 
##        320267        237782         20931         16428         20181 
##  3.628882e+01  3.625349e+01  3.613864e+01  3.608904e+01  3.603215e+01 
##         68859         20687        226178         55947         70024 
##  3.598548e+01  3.586973e+01  3.574848e+01  3.573102e+01  3.563725e+01 
##        332397        216856     100043272        140481         67187 
##  3.563109e+01  3.545899e+01  3.542441e+01  3.542132e+01  3.517144e+01 
##        242705         64138        217125         30839         75146 
##  3.514481e+01  3.501378e+01  3.495877e+01  3.491388e+01  3.483478e+01 
##         19224         18646         20498         54486        212706 
##  3.467978e+01  3.457069e+01  3.456742e+01  3.453417e+01  3.443767e+01 
##        226122        228536         56807         14582        226139 
##  3.443156e+01  3.434003e+01  3.433655e+01  3.431437e+01  3.425673e+01 
##         15451         11909         15957         67337         14461 
##  3.422002e+01  3.405944e+01  3.395343e+01  3.395243e+01  3.391995e+01 
##        140570         13667         11841        100764        226154 
##  3.390969e+01  3.389900e+01  3.375010e+01  3.364950e+01  3.364702e+01 
##        239849         98956        213993         53608        110454 
##  3.357202e+01  3.354549e+01  3.353459e+01  3.350855e+01  3.347452e+01 
##        110911        433638         67073        118451         14538 
##  3.345585e+01  3.343617e+01  3.343282e+01  3.337401e+01  3.335549e+01 
##         17082        382062         12751        269639         13807 
##  3.329699e+01  3.325615e+01  3.318406e+01  3.315776e+01  3.303291e+01 
##        232970         54338         14728         11790         12581 
##  3.298517e+01  3.291139e+01  3.256546e+01  3.254725e+01  3.252557e+01 
##         68968         27214         16391         80985         56046 
##  3.250934e+01  3.246071e+01  3.231419e+01  3.217800e+01  3.212738e+01 
##         11432         16418        241303         67116         66960 
##  3.209647e+01  3.209337e+01  3.209019e+01  3.207956e+01  3.203267e+01 
##         56336         20256        263876        330627         75723 
##  3.203069e+01  3.202733e+01  3.202182e+01  3.200370e+01  3.179540e+01 
##         73737         17858        227656         52357         72399 
##  3.156118e+01  3.155513e+01  3.148830e+01  3.147153e+01  3.145150e+01 
##         22030         12568         54130         19303         66390 
##  3.138772e+01  3.136411e+01  3.132189e+01  3.132186e+01  3.127536e+01 
##        100910         77832         22325        231633         13537 
##  3.111159e+01  3.094578e+01  3.093229e+01  3.092735e+01  3.081019e+01 
##        209027         14739         26373         68774        117150 
##  3.080159e+01  3.066605e+01  3.057093e+01  3.053887e+01  3.053260e+01 
##         21349         54711        109079         11519         65103 
##  3.045365e+01  3.030003e+01  3.025799e+01  3.023212e+01  3.020864e+01 
##         16449        227715         77590         15211        230073 
##  3.017782e+01  3.010069e+01  3.003212e+01  2.997833e+01  2.991566e+01 
##         67204         81018        109275         66366        227648 
##  2.982324e+01  2.977419e+01  2.971673e+01  2.971496e+01  2.966749e+01 
##         12589         56317        228368         66404         58238 
##  2.966385e+01  2.958669e+01  2.954877e+01  2.945823e+01  2.943131e+01 
##        228889         20304         81910        215707         80751 
##  2.940952e+01  2.927426e+01  2.923871e+01  2.922893e+01  2.921343e+01 
##        319513        228852        109129         16411     100038882 
##  2.906222e+01  2.905666e+01  2.897698e+01  2.890503e+01  2.883950e+01 
##         56406         68379         74451        215632         78890 
##  2.880943e+01  2.879337e+01  2.870648e+01  2.858741e+01  2.858028e+01 
##         19247         72542        209630        231070         70769 
##  2.857376e+01  2.856212e+01  2.850590e+01  2.846370e+01  2.839376e+01 
##         56715        320951         72080        227659         18572 
##  2.822388e+01  2.820170e+01  2.813303e+01  2.810940e+01  2.809298e+01 
##         76220        239273         20935        233016         75608 
##  2.807088e+01  2.806256e+01  2.803893e+01  2.781411e+01  2.768046e+01 
##         57170         20963         12369        672511         23992 
##  2.764660e+01  2.761271e+01  2.759614e+01  2.758702e+01  2.758231e+01 
##         56398         68732         66234         80743         80909 
##  2.755620e+01  2.744973e+01  2.743551e+01  2.743355e+01  2.742823e+01 
##         77573        108682         73373        407243         58185 
##  2.742095e+01  2.737174e+01  2.723500e+01  2.721844e+01  2.715463e+01 
##         77038         80290         67870         18984         74168 
##  2.709473e+01  2.696226e+01  2.695819e+01  2.691601e+01  2.691072e+01 
##        227644         22034         68842         13486         18590 
##  2.690538e+01  2.686410e+01  2.686397e+01  2.684433e+01  2.679514e+01 
##         76501         12772        217866        209361         19106 
##  2.678040e+01  2.678028e+01  2.673601e+01  2.673398e+01  2.671836e+01 
##         14208        215653        252972         67382         70335 
##  2.667828e+01  2.665091e+01  2.656558e+01  2.649492e+01  2.645125e+01 
##        101631        227720         12615        227613         68295 
##  2.644196e+01  2.639402e+01  2.632357e+01  2.629771e+01  2.628955e+01 
##         18457         75786         71715         76792         20540 
##  2.628435e+01  2.627338e+01  2.626024e+01  2.625662e+01  2.624361e+01 
##         72119         23790         68112         72199        433926 
##  2.622325e+01  2.620842e+01  2.617988e+01  2.617008e+01  2.616227e+01 
##        330064         74777         66168         20833        209131 
##  2.606192e+01  2.606014e+01  2.602675e+01  2.602578e+01  2.601183e+01 
##        215387         99326        226169         83814         68475 
##  2.598775e+01  2.597388e+01  2.594267e+01  2.588590e+01  2.585574e+01 
##     100503392         69185        331474         72326        227638 
##  2.584305e+01  2.583414e+01  2.573718e+01  2.567720e+01  2.567520e+01 
##         74136        227697         56876         17330         19181 
##  2.566051e+01  2.561509e+01  2.561440e+01  2.560011e+01  2.559287e+01 
##        140780        171285         11502         68375         30838 
##  2.545949e+01  2.542204e+01  2.540904e+01  2.540542e+01  2.537486e+01 
##         74498         13805         72477         68059         67956 
##  2.532298e+01  2.531992e+01  2.523034e+01  2.521952e+01  2.518385e+01 
##         68098         64436         68948        231600         70239 
##  2.513231e+01  2.511925e+01  2.511397e+01  2.494660e+01  2.494159e+01 
##        320827         72075        107338         51885         11480 
##  2.489946e+01  2.486358e+01  2.482350e+01  2.480758e+01  2.474859e+01 
##         58206         20692         14788     100502982         73847 
##  2.474547e+01  2.473237e+01  2.472955e+01  2.469459e+01  2.467695e+01 
##         74528         53893        106068         19667        228061 
##  2.467687e+01  2.466039e+01  2.465330e+01  2.464581e+01  2.462047e+01 
##         66583        227731         77683         80291        382018 
##  2.458578e+01  2.457818e+01  2.452539e+01  2.447454e+01  2.444079e+01 
##        110391         75665        109305         93961         26457 
##  2.441536e+01  2.440690e+01  2.436111e+01  2.431355e+01  2.421660e+01 
##        228769         14810         20619         67865        269608 
##  2.420289e+01  2.417732e+01  2.401143e+01  2.398252e+01  2.393562e+01 
##        231506         50935         77938         69534         56422 
##  2.387531e+01  2.384794e+01  2.384455e+01  2.383236e+01  2.382033e+01 
##         99334         71111         13429         11949        319371 
##  2.381810e+01  2.375280e+01  2.371801e+01  2.370265e+01  2.347559e+01 
##         53310         21982         68695         60525         27784 
##  2.345178e+01  2.344675e+01  2.341015e+01  2.336662e+01  2.336650e+01 
##        231915         99138         66395         15937        216835 
##  2.336149e+01  2.336027e+01  2.335950e+01  2.333246e+01  2.331914e+01 
##         20514        231655         56048         19664        671392 
##  2.325466e+01  2.323937e+01  2.323717e+01  2.321407e+01  2.316728e+01 
##        228482        109299         66300         76467        320717 
##  2.313500e+01  2.306872e+01  2.306624e+01  2.303580e+01  2.299073e+01 
##         98221        381410        211499         53890         12571 
##  2.298862e+01  2.297640e+01  2.295480e+01  2.295409e+01  2.283385e+01 
##         18796         70021        233066         97031        242864 
##  2.278414e+01  2.273925e+01  2.269214e+01  2.264533e+01  2.264081e+01 
##         74270         18081         76809         52064         21664 
##  2.263639e+01  2.254313e+01  2.254136e+01  2.253549e+01  2.252980e+01 
##         14287         68480         68795        231130         66998 
##  2.252836e+01  2.248388e+01  2.247522e+01  2.240601e+01  2.239168e+01 
##         21928         68201         72649         59003        234407 
##  2.234954e+01  2.234072e+01  2.229603e+01  2.226856e+01  2.225893e+01 
##         70231         12217         74254         20014         67059 
##  2.222661e+01  2.222130e+01  2.217897e+01  2.214315e+01  2.209571e+01 
##         52666         56428         12267         77006         72543 
##  2.206537e+01  2.196624e+01  2.179349e+01  2.176854e+01  2.174757e+01 
##         16396        110854         20317        106952         19206 
##  2.173889e+01  2.173672e+01  2.172623e+01  2.172033e+01  2.171754e+01 
##         99151         66711         24116        243374        381066 
##  2.171363e+01  2.169351e+01  2.158918e+01  2.158554e+01  2.158462e+01 
##         56470        107733         12235        666060         75416 
##  2.148075e+01  2.147871e+01  2.142521e+01  2.134746e+01  2.129797e+01 
##         70744        330177         13014         11958         66296 
##  2.128703e+01  2.124283e+01  2.122991e+01  2.122843e+01  2.122146e+01 
##         17748         20847        330256         58202         22340 
##  2.121680e+01  2.120704e+01  2.118530e+01  2.117509e+01  2.116399e+01 
##         73024     100041194         27008        243961        330192 
##  2.115010e+01  2.110508e+01  2.106423e+01  2.105828e+01  2.104649e+01 
##         73658        140484        664888        170768         16923 
##  2.100186e+01  2.096308e+01  2.094596e+01  2.093063e+01  2.091406e+01 
##        209039         74504         56075         69719         68796 
##  2.089390e+01  2.081941e+01  2.080642e+01  2.070822e+01  2.069400e+01 
##         30931         16452         18516         20844        231769 
##  2.065966e+01  2.065073e+01  2.059681e+01  2.056730e+01  2.053378e+01 
##         77697         20128         83493     100503839         67126 
##  2.052494e+01  2.051817e+01  2.048748e+01  2.045884e+01  2.044771e+01 
##         19400         27261        224674         94219        216161 
##  2.042487e+01  2.041130e+01  2.035537e+01  2.035139e+01  2.033126e+01 
##         99011        269630         66559         66866         56324 
##  2.023303e+01  2.021028e+01  2.018578e+01  2.018462e+01  2.017877e+01 
##         80285         56407         66587        107976        231151 
##  2.013206e+01  2.012871e+01  2.009280e+01  2.004529e+01  2.001784e+01 
##        140579         56491        214616        207565        240613 
##  2.000409e+01  1.999501e+01  1.994940e+01  1.991725e+01  1.988777e+01 
##         53978         54447        227683         29816        209357 
##  1.987670e+01  1.986024e+01  1.984465e+01  1.973686e+01  1.972675e+01 
##         57743         19731        243780        242860         14460 
##  1.969343e+01  1.964759e+01  1.963920e+01  1.963726e+01  1.963412e+01 
##         74349         13418        100756         18538         12909 
##  1.960181e+01  1.956970e+01  1.956477e+01  1.956092e+01  1.955864e+01 
##        100737         69976        208967        140629        192156 
##  1.950016e+01  1.946584e+01  1.946014e+01  1.943209e+01  1.941510e+01 
##         64707        227723        195040         19730        246696 
##  1.941448e+01  1.931106e+01  1.930353e+01  1.928412e+01  1.925723e+01 
##         22130        108958        227522         14998        223920 
##  1.924214e+01  1.923441e+01  1.919739e+01  1.918288e+01  1.915772e+01 
##         16783         94254         69202         77407         71971 
##  1.914220e+01  1.910511e+01  1.905802e+01  1.905480e+01  1.902644e+01 
##        212772         13011         12234         66170        227525 
##  1.900513e+01  1.899165e+01  1.896819e+01  1.894715e+01  1.892640e+01 
##         64945        330189        215210         27402         56017 
##  1.890844e+01  1.886513e+01  1.885177e+01  1.884320e+01  1.882776e+01 
##        209086        228033         54426         12545         66679 
##  1.879774e+01  1.878088e+01  1.877252e+01  1.875141e+01  1.874527e+01 
##         13828         16477        109689         71834        226252 
##  1.874362e+01  1.873975e+01  1.873030e+01  1.872419e+01  1.864676e+01 
##         66258         22791         19054         73102        227674 
##  1.862418e+01  1.862009e+01  1.861799e+01  1.855086e+01  1.851878e+01 
##        319740         56086         20930        654470     100041407 
##  1.850906e+01  1.849193e+01  1.847712e+01  1.846793e+01  1.846765e+01 
##         27267         74243         14605         66848        217344 
##  1.841043e+01  1.838966e+01  1.834897e+01  1.833682e+01  1.833222e+01 
##         98766         14854         67182        225849         53611 
##  1.833167e+01  1.831582e+01  1.827593e+01  1.827043e+01  1.826425e+01 
##         27054        226182         67388         11834         67122 
##  1.822693e+01  1.822460e+01  1.820784e+01  1.818049e+01  1.817563e+01 
##         66934     100039636        101023         67333         69104 
##  1.810365e+01  1.810276e+01  1.800746e+01  1.800024e+01  1.799601e+01 
##         57915        223255         69605         66980         66958 
##  1.797754e+01  1.795390e+01  1.793421e+01  1.790318e+01  1.787742e+01 
##         71667         20226        228357         52392         66169 
##  1.787119e+01  1.783127e+01  1.783001e+01  1.782435e+01  1.779987e+01 
##         56378         52013         56488         50765         69895 
##  1.778193e+01  1.775386e+01  1.773896e+01  1.773356e+01  1.771454e+01 
##         67752         19246         71820         56480         71085 
##  1.771303e+01  1.770237e+01  1.768473e+01  1.767329e+01  1.766477e+01 
##        330812         19015         27377        102423         94279 
##  1.766391e+01  1.765898e+01  1.763543e+01  1.762808e+01  1.762723e+01 
##         71720         57314         77799        215280         18189 
##  1.762625e+01  1.761071e+01  1.759046e+01  1.751974e+01  1.749555e+01 
##        226144         66141         78894         66442         98828 
##  1.748791e+01  1.739349e+01  1.737350e+01  1.737229e+01  1.734723e+01 
##         20602         15042         66602         26429        217684 
##  1.733821e+01  1.732587e+01  1.731542e+01  1.730565e+01  1.728253e+01 
##         16434        105348        381650        381644         72931 
##  1.726806e+01  1.726572e+01  1.722666e+01  1.718985e+01  1.718724e+01 
##        381379         27176         12476        228715         72008 
##  1.714424e+01  1.712777e+01  1.709465e+01  1.705948e+01  1.703125e+01 
##        241633        227738         50907        320415         68949 
##  1.702987e+01  1.702363e+01  1.701826e+01  1.700635e+01  1.699547e+01 
##         54396         12454        208084        231713         69113 
##  1.699079e+01  1.698895e+01  1.696538e+01  1.693352e+01  1.693331e+01 
##         68836         12908        268749         19383        192236 
##  1.692852e+01  1.691922e+01  1.689209e+01  1.688586e+01  1.687082e+01 
##         15194         17095         20972         71752         19744 
##  1.686642e+01  1.684715e+01  1.684248e+01  1.683485e+01  1.677055e+01 
##         76824        228019        240665         11350         14571 
##  1.677015e+01  1.676485e+01  1.674457e+01  1.672371e+01  1.671081e+01 
##        228998        329679         58220         67905         13852 
##  1.670804e+01  1.666615e+01  1.666262e+01  1.658853e+01  1.658327e+01 
##        259300         75894         12051         72146         12700 
##  1.656865e+01  1.654867e+01  1.654339e+01  1.653285e+01  1.652631e+01 
##         20556         11308         26433         76491        545878 
##  1.643243e+01  1.642400e+01  1.641418e+01  1.635954e+01  1.633394e+01 
##         13435         66617         16328        319801         69470 
##  1.629228e+01  1.628438e+01  1.625220e+01  1.623990e+01  1.623736e+01 
##         14528        228850         23961         50497        228961 
##  1.619213e+01  1.617624e+01  1.617522e+01  1.614866e+01  1.613693e+01 
##        192157         75763         98910         66905         19725 
##  1.612733e+01  1.609988e+01  1.606529e+01  1.604568e+01  1.599504e+01 
##        433144     102633081         17874        191578         22761 
##  1.599343e+01  1.598964e+01  1.591621e+01  1.589383e+01  1.588743e+01 
##         68612         68925         16825         12995        326618 
##  1.588574e+01  1.588103e+01  1.588089e+01  1.587373e+01  1.586907e+01 
##         56013         12332         70408         68082         11911 
##  1.585957e+01  1.584212e+01  1.583719e+01  1.582398e+01  1.580643e+01 
##         16987         13010        327978         99100         12848 
##  1.580085e+01  1.579442e+01  1.576975e+01  1.576741e+01  1.575577e+01 
##         68929         21353         30930         74178         66460 
##  1.575107e+01  1.574061e+01  1.572983e+01  1.571658e+01  1.571303e+01 
##        667618        399568        227624        107526        277396 
##  1.570799e+01  1.569240e+01  1.568538e+01  1.567811e+01  1.565759e+01 
##         72425         69082         13033        100017         53323 
##  1.563906e+01  1.560670e+01  1.557335e+01  1.557090e+01  1.554106e+01 
##         70567         66528         24069         76688         18108 
##  1.553172e+01  1.552519e+01  1.551968e+01  1.550545e+01  1.547968e+01 
##         79464         12728         66044         19201        226162 
##  1.546202e+01  1.545879e+01  1.540065e+01  1.539685e+01  1.538010e+01 
##         30050        269704         68365         72657         68080 
##  1.537226e+01  1.535396e+01  1.534518e+01  1.534496e+01  1.531395e+01 
##        224617         83410        269717         19262        243963 
##  1.528425e+01  1.527290e+01  1.524421e+01  1.520700e+01  1.520109e+01 
##         73338        100978        320720         15980        170741 
##  1.519652e+01  1.518263e+01  1.514064e+01  1.513296e+01  1.509992e+01 
##         12753         19822         78885        353346        241308 
##  1.507896e+01  1.506453e+01  1.502719e+01  1.501984e+01  1.501830e+01 
##         13423         14387     100042480        665489         59024 
##  1.501278e+01  1.499252e+01  1.497800e+01  1.493895e+01  1.487755e+01 
##         67568         17713        100855         71782         76246 
##  1.487068e+01  1.485930e+01  1.485565e+01  1.485453e+01  1.483211e+01 
##        320790         59092        214048         30791         14718 
##  1.482335e+01  1.481272e+01  1.479244e+01  1.477853e+01  1.477006e+01 
##     100042823        194268         99045         66627         66374 
##  1.476083e+01  1.475503e+01  1.474864e+01  1.471164e+01  1.469091e+01 
##        381629         27428         12445        229004         14466 
##  1.468386e+01  1.467852e+01  1.463637e+01  1.462037e+01  1.461624e+01 
##         17937        228005         65107         71990         17134 
##  1.461235e+01  1.461031e+01  1.460970e+01  1.460879e+01  1.459249e+01 
##         11674         72057         67291         56433         71729 
##  1.458335e+01  1.457919e+01  1.457225e+01  1.449618e+01  1.448015e+01 
##         56361         67469         12675         67231         18174 
##  1.445498e+01  1.441907e+01  1.441202e+01  1.440742e+01  1.438900e+01 
##         55984        319939         66383         11610         66140 
##  1.434501e+01  1.434155e+01  1.433317e+01  1.433226e+01  1.430737e+01 
##         16590         57444        107951     115490131        245666 
##  1.429176e+01  1.426467e+01  1.423324e+01  1.422071e+01  1.417237e+01 
##        269336         68975         76355         51897         68659 
##  1.417163e+01  1.416672e+01  1.416108e+01  1.415764e+01  1.412791e+01 
##        212073         56384         52856         11800        380711 
##  1.408497e+01  1.402967e+01  1.398747e+01  1.398288e+01  1.397540e+01 
##         20778         12505         80708         69550        233902 
##  1.396876e+01  1.394563e+01  1.390136e+01  1.389705e+01  1.388141e+01 
##         11776         76080         66988         73822         67228 
##  1.386768e+01  1.385187e+01  1.384941e+01  1.383061e+01  1.382992e+01 
##         12322         67695        329540        209446         23882 
##  1.381556e+01  1.381462e+01  1.377206e+01  1.377132e+01  1.375926e+01 
##         11990         74533         56626         13401         71972 
##  1.375793e+01  1.373232e+01  1.371221e+01  1.370551e+01  1.370422e+01 
##         72058        100608         56699        117160         12802 
##  1.369786e+01  1.368621e+01  1.368048e+01  1.366072e+01  1.363970e+01 
##         11987        241950        212090         67136        104859 
##  1.363606e+01  1.362784e+01  1.357909e+01  1.357018e+01  1.355119e+01 
##         17974        192232         20359        108014         71772 
##  1.354202e+01  1.353515e+01  1.352713e+01  1.350957e+01  1.349512e+01 
##         83767        228994        328833         97775        268515 
##  1.347368e+01  1.346056e+01  1.343649e+01  1.338661e+01  1.338591e+01 
##        216134         11490         66999        231659         71041 
##  1.338115e+01  1.337339e+01  1.333804e+01  1.332791e+01  1.331955e+01 
##         78796         66181         13496     100042651         98985 
##  1.330843e+01  1.330755e+01  1.328964e+01  1.327835e+01  1.327134e+01 
##         67894         58250     102636251         68215         19988 
##  1.325267e+01  1.323130e+01  1.322627e+01  1.321761e+01  1.319615e+01 
##         83435         11542        104027        266781         22343 
##  1.318243e+01  1.317425e+01  1.315986e+01  1.312205e+01  1.308625e+01 
##     100504029         67848        381353         74111         58240 
##  1.306983e+01  1.304726e+01  1.302107e+01  1.301311e+01  1.301177e+01 
##         19211         64930        231991        231672        269400 
##  1.299251e+01  1.298992e+01  1.298801e+01  1.298284e+01  1.296384e+01 
##        107435        228812        381668         17151         67878 
##  1.296269e+01  1.293916e+01  1.292954e+01  1.292771e+01  1.292232e+01 
##         70612         74268         14269        228790         12396 
##  1.291751e+01  1.291361e+01  1.291099e+01  1.290558e+01  1.289880e+01 
##         20411     105244102         56369         66314        246730 
##  1.289553e+01  1.289000e+01  1.287337e+01  1.285265e+01  1.283902e+01 
##         29870        433931     100417946         13121        224807 
##  1.283287e+01  1.282459e+01  1.278379e+01  1.277545e+01  1.277465e+01 
##         14431         67886     100040331        241694        213948 
##  1.276094e+01  1.275803e+01  1.273777e+01  1.273257e+01  1.270405e+01 
##         20852        107358        320024         52398         75735 
##  1.267819e+01  1.265951e+01  1.265913e+01  1.265793e+01  1.264158e+01 
##        226089        231225        226151         13830        231128 
##  1.255353e+01  1.252201e+01  1.252019e+01  1.251964e+01  1.251798e+01 
##         17863         69727         16478        100763        212999 
##  1.251159e+01  1.250815e+01  1.249461e+01  1.249126e+01  1.248849e+01 
##        109241        214585         18508         74309        269610 
##  1.248847e+01  1.248039e+01  1.247446e+01  1.244902e+01  1.243927e+01 
##         72357        100465        627624         66461        105844 
##  1.243913e+01  1.243774e+01  1.243100e+01  1.242723e+01  1.241081e+01 
##         69707         69663         66548         56348         66632 
##  1.241051e+01  1.240924e+01  1.240857e+01  1.240172e+01  1.234191e+01 
##         80744         16365         75812         21788         26556 
##  1.233996e+01  1.233299e+01  1.232263e+01  1.231947e+01  1.230736e+01 
##         18030     102633666        109711         67857        231580 
##  1.230647e+01  1.230114e+01  1.229996e+01  1.225996e+01  1.222004e+01 
##         11433        107173        227707         16768        140571 
##  1.221802e+01  1.221484e+01  1.219597e+01  1.218183e+01  1.217123e+01 
##         70387         20339        433204         15357         83945 
##  1.215909e+01  1.211955e+01  1.210253e+01  1.208303e+01  1.206462e+01 
##        277973         58245        110524         11757         74167 
##  1.206224e+01  1.204399e+01  1.202880e+01  1.200588e+01  1.197684e+01 
##         13857         18540        381678        227753         18442 
##  1.195289e+01  1.194380e+01  1.191589e+01  1.187389e+01  1.186631e+01 
##        228602         69635         97487        209354         16410 
##  1.183916e+01  1.183376e+01  1.182455e+01  1.182141e+01  1.180683e+01 
##        107271         68108         67871        320816         66812 
##  1.178626e+01  1.178043e+01  1.177872e+01  1.177165e+01  1.176290e+01 
##         27762         72309         14537         19334         74919 
##  1.176267e+01  1.172883e+01  1.169175e+01  1.169078e+01  1.168776e+01 
##        228413        217935        216459         17765         66634 
##  1.168430e+01  1.166631e+01  1.166359e+01  1.166195e+01  1.164587e+01 
##         19079         68328         70584         54125         79264 
##  1.163599e+01  1.163234e+01  1.163232e+01  1.162943e+01  1.160692e+01 
##         74585         74159        231670         18515        233890 
##  1.160337e+01  1.159614e+01  1.157107e+01  1.156849e+01  1.155725e+01 
##         57908         24055         67068         69178         13589 
##  1.153521e+01  1.153467e+01  1.150769e+01  1.149918e+01  1.149348e+01 
##         52690        328162        227743         56068         74239 
##  1.146194e+01  1.145405e+01  1.141896e+01  1.140332e+01  1.139390e+01 
##         57442         71382         67630         70572         19401 
##  1.138122e+01  1.137239e+01  1.137181e+01  1.134595e+01  1.134449e+01 
##        108160         13016     102637961         70556     119065974 
##  1.134178e+01  1.133764e+01  1.133478e+01  1.133159e+01  1.132447e+01 
##         74105         66481        240641        107823         66335 
##  1.132197e+01  1.131284e+01  1.131116e+01  1.130599e+01  1.129620e+01 
##         28169         71838         76192        433864         14773 
##  1.129430e+01  1.126617e+01  1.125940e+01  1.125783e+01  1.124924e+01 
##        381903         56350        223739        226016         12494 
##  1.124018e+01  1.122565e+01  1.120974e+01  1.117856e+01  1.116498e+01 
##        231855         19743         13669        226251         11615 
##  1.113448e+01  1.112869e+01  1.112420e+01  1.112292e+01  1.112130e+01 
##         67759         66801        114585        227699         71617 
##  1.110743e+01  1.109986e+01  1.109265e+01  1.106952e+01  1.104919e+01 
##         72727         20535        114143         66940         50849 
##  1.098885e+01  1.097851e+01  1.096144e+01  1.096001e+01  1.094790e+01 
##        231452         13026         11837         72502         67194 
##  1.094361e+01  1.093845e+01  1.093750e+01  1.092979e+01  1.092532e+01 
##        212555        229007         17200         53312        109202 
##  1.092089e+01  1.090944e+01  1.090211e+01  1.089745e+01  1.087919e+01 
##        233765         69987        107272        675812         66939 
##  1.087644e+01  1.086147e+01  1.083979e+01  1.083151e+01  1.082959e+01 
##         78412         68095         12611         20170         69553 
##  1.080570e+01  1.079789e+01  1.078573e+01  1.078367e+01  1.078092e+01 
##         66586         12521         72621         17342        320292 
##  1.077017e+01  1.076012e+01  1.073567e+01  1.073421e+01  1.073300e+01 
##        228361         71954        241627         14284        231571 
##  1.070740e+01  1.068408e+01  1.067043e+01  1.064252e+01  1.063319e+01 
##        320187        278279         22245        433693        269700 
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##         12562         67809        212919        116873         64704 
##  1.061005e+01  1.060950e+01  1.058729e+01  1.057927e+01  1.057692e+01 
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##  1.057037e+01  1.056082e+01  1.055270e+01  1.055069e+01  1.054274e+01 
##         22029         30963     100042807         58231         60533 
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##  1.042967e+01  1.040229e+01  1.039615e+01  1.038458e+01  1.038134e+01 
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##  1.036812e+01  1.034645e+01  1.033169e+01  1.033065e+01  1.031542e+01 
##        432466         68889        243300         12497         15424 
##  1.030987e+01  1.030879e+01  1.030168e+01  1.029371e+01  1.029138e+01 
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##  1.028041e+01  1.027463e+01  1.025672e+01  1.025640e+01  1.024863e+01 
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##  1.024739e+01  1.023674e+01  1.023105e+01  1.022358e+01  1.021090e+01 
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##  1.020179e+01  1.019857e+01  1.018030e+01  1.017782e+01  1.016804e+01 
##         76464         78926        626000         99371        227867 
##  1.016714e+01  1.014887e+01  1.013455e+01  1.012332e+01  1.011432e+01 
##        435684         93960        105663         26431         74251 
##  1.010400e+01  1.009712e+01  1.009321e+01  1.008660e+01  1.007504e+01 
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##  1.006108e+01  1.004802e+01  1.004202e+01  1.003333e+01  1.001633e+01 
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##     100037258         18761        320878         74412         21335 
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##  9.419302e+00  9.391781e+00  9.367374e+00  9.366107e+00  9.361804e+00 
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##  9.139271e+00  9.122877e+00  9.121346e+00  9.106656e+00  9.105028e+00 
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##  8.999656e+00  8.995178e+00  8.994345e+00  8.979027e+00  8.978982e+00 
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##  8.927381e+00  8.924967e+00  8.924498e+00  8.923487e+00  8.921666e+00 
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##  8.901473e+00  8.897652e+00  8.896234e+00  8.885990e+00  8.883637e+00 
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##  8.855831e+00  8.854073e+00  8.843908e+00  8.826387e+00  8.823869e+00 
##         13875        319622         83679         19361         72572 
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##  8.603709e+00  8.593821e+00  8.593334e+00  8.578427e+00  8.561353e+00 
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##  8.106685e+00  8.098831e+00  8.094046e+00  8.069498e+00  8.065089e+00 
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##  8.060361e+00  8.046785e+00  8.043441e+00  8.043197e+00  8.043148e+00 
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##  6.246841e+00  6.243681e+00  6.242529e+00  6.240455e+00  6.239459e+00 
##         58800         57443        260299        665536         76608 
##  6.230292e+00  6.217605e+00  6.206098e+00  6.204811e+00  6.198426e+00 
##        233033        228550        434076     100689703         19039 
##  6.191931e+00  6.185964e+00  6.185038e+00  6.184521e+00  6.183447e+00 
##         68879         18770         66333         57753        100561 
##  6.181876e+00  6.170278e+00  6.165877e+00  6.161105e+00  6.156421e+00 
##         18207         74455        210009         68332     100043133 
##  6.154679e+00  6.145846e+00  6.144791e+00  6.140399e+00  6.124869e+00 
##         12585         56395        241624         14347        319772 
##  6.123597e+00  6.118935e+00  6.112118e+00  6.111987e+00  6.106574e+00 
##        230098        330171        109229        104215         22632 
##  6.106183e+00  6.099383e+00  6.088857e+00  6.088739e+00  6.088427e+00 
##        107817        244666         21425         20467         56327 
##  6.084730e+00  6.084296e+00  6.080494e+00  6.075694e+00  6.075574e+00 
##        100177        666266         20909        104718         78697 
##  6.052048e+00  6.050919e+00  6.050032e+00  6.029238e+00  6.029210e+00 
##        215194         14693        545592         16548         26395 
##  6.027256e+00  6.025115e+00  6.014304e+00  6.006034e+00  6.002202e+00 
##     102638850        107368        223433         18753        215474 
##  5.993944e+00  5.990582e+00  5.988768e+00  5.978431e+00  5.976830e+00 
##        207175        210004         71839         59047         12453 
##  5.964366e+00  5.960424e+00  5.960132e+00  5.955336e+00  5.950487e+00 
##        214575        232086        208366        241846        208431 
##  5.948943e+00  5.948367e+00  5.947476e+00  5.941900e+00  5.936684e+00 
##         18378         20439     100502698        211401         67803 
##  5.936314e+00  5.933483e+00  5.931255e+00  5.927222e+00  5.924278e+00 
##        243272        208266        231807         16421         73062 
##  5.913898e+00  5.906892e+00  5.906409e+00  5.901771e+00  5.897275e+00 
##         76969         15547         52348         66143         67078 
##  5.896517e+00  5.892509e+00  5.892390e+00  5.886948e+00  5.883230e+00 
##         12593         12981     100039316        100494     100043022 
##  5.875753e+00  5.871641e+00  5.864921e+00  5.863565e+00  5.862397e+00 
##         69596         67588         22214         77613         75423 
##  5.850146e+00  5.839951e+00  5.837373e+00  5.835141e+00  5.813780e+00 
##        215705         69482         56455         27029        434218 
##  5.805665e+00  5.804604e+00  5.803925e+00  5.798800e+00  5.797289e+00 
##        223658         14789         72472         66580         69993 
##  5.796005e+00  5.794804e+00  5.788901e+00  5.785398e+00  5.776485e+00 
##         17089        414758         70103         18704         15356 
##  5.772746e+00  5.766646e+00  5.765113e+00  5.765108e+00  5.762199e+00 
##         71881        269529         18601        216150     100504461 
##  5.755889e+00  5.754790e+00  5.731996e+00  5.728846e+00  5.728681e+00 
##         11898        269623         12795        227721         73571 
##  5.723703e+00  5.714943e+00  5.714277e+00  5.700495e+00  5.700045e+00 
##        666465        667264         11867        320879         66388 
##  5.699731e+00  5.699719e+00  5.688830e+00  5.686400e+00  5.684982e+00 
##        329384         70495         22193     102631836         26427 
##  5.684146e+00  5.676539e+00  5.667240e+00  5.659458e+00  5.656532e+00 
##         67804        140859         14283         14104     105246278 
##  5.653012e+00  5.652930e+00  5.651381e+00  5.648804e+00  5.647879e+00 
##        213753        624438         12861         69109         60611 
##  5.647470e+00  5.637982e+00  5.629148e+00  5.625463e+00  5.617893e+00 
##        108011         83490        194952        399627         22222 
##  5.616943e+00  5.616587e+00  5.615450e+00  5.614310e+00  5.613777e+00 
##         56412         68837         14885        209478        270198 
##  5.601462e+00  5.601271e+00  5.584589e+00  5.581354e+00  5.576163e+00 
##        117589         67211         52668        228829         71116 
##  5.575861e+00  5.569613e+00  5.566007e+00  5.562620e+00  5.560873e+00 
##     100043424        108961         52504        665775     115488793 
##  5.556510e+00  5.546368e+00  5.540078e+00  5.533334e+00  5.532847e+00 
##         18973        102448         75079         72775        629378 
##  5.518723e+00  5.517876e+00  5.516397e+00  5.513453e+00  5.510463e+00 
##        110460         57914         20163         56401         11845 
##  5.506896e+00  5.506642e+00  5.499055e+00  5.483886e+00  5.478169e+00 
##         70223        213499        434402        668943         70417 
##  5.477916e+00  5.477449e+00  5.467260e+00  5.463944e+00  5.461385e+00 
##         76199         67087         66661        235584        232406 
##  5.455816e+00  5.455419e+00  5.448925e+00  5.446539e+00  5.435810e+00 
##        217887        116904        217364         20775         73137 
##  5.435115e+00  5.431540e+00  5.424224e+00  5.417941e+00  5.413701e+00 
##         17192         16453         12977         16784         55982 
##  5.412253e+00  5.412074e+00  5.406333e+00  5.402802e+00  5.397828e+00 
##        216443        195522         13605         24001         14950 
##  5.395917e+00  5.395122e+00  5.394679e+00  5.390525e+00  5.385181e+00 
##        228859        170826         56736         21957         68385 
##  5.384593e+00  5.381202e+00  5.380754e+00  5.375729e+00  5.373909e+00 
##         67877        407803         14999         12492        235461 
##  5.370995e+00  5.364102e+00  5.360640e+00  5.353678e+00  5.330393e+00 
##         20853         70381         19183        319710         75991 
##  5.330387e+00  5.329816e+00  5.324878e+00  5.319741e+00  5.319360e+00 
##        234839         16542        407819         30057         67350 
##  5.313382e+00  5.303085e+00  5.291423e+00  5.273747e+00  5.267685e+00 
##         50868         16153        268882        630138         13006 
##  5.267426e+00  5.261687e+00  5.260293e+00  5.256146e+00  5.254194e+00 
##         18824        216119        433408        224840        170756 
##  5.248240e+00  5.241319e+00  5.226941e+00  5.223261e+00  5.221544e+00 
##     108169069        381232         76100         66839        625958 
##  5.215092e+00  5.212650e+00  5.208321e+00  5.200104e+00  5.190440e+00 
##        269061         12009         67732        235406        245622 
##  5.186617e+00  5.186454e+00  5.183890e+00  5.183479e+00  5.181451e+00 
##        110816         75869        665829         68048         79560 
##  5.171092e+00  5.169510e+00  5.167196e+00  5.166394e+00  5.163551e+00 
##        208449        667606         75296        268319        625054 
##  5.162788e+00  5.155660e+00  5.145744e+00  5.144208e+00  5.143820e+00 
##        210992        106326     105244803        320806         77853 
##  5.139484e+00  5.138259e+00  5.137702e+00  5.136707e+00  5.135515e+00 
##        246746         22375        211488        100532        385674 
##  5.135078e+00  5.134761e+00  5.129988e+00  5.122217e+00  5.121388e+00 
##         66236         23980         52857        380732         59005 
##  5.087695e+00  5.085687e+00  5.075567e+00  5.074863e+00  5.074040e+00 
##         58996        106759        276852         68728         67121 
##  5.073065e+00  5.071003e+00  5.068589e+00  5.068342e+00  5.067432e+00 
##         17714         71943     102632299         68170         67554 
##  5.065207e+00  5.061720e+00  5.060289e+00  5.057778e+00  5.042970e+00 
##         74596        219144        233905         76375        207952 
##  5.022412e+00  5.019702e+00  5.018855e+00  5.016171e+00  5.013231e+00 
##        100163        208638     102635008         21886        320271 
##  5.007432e+00  5.007059e+00  4.994770e+00  4.991704e+00  4.984859e+00 
##         27360         16443         69743        228410         65247 
##  4.975729e+00  4.972100e+00  4.967522e+00  4.964364e+00  4.957945e+00 
##        231841        319594         70122        213081         15982 
##  4.951353e+00  4.949278e+00  4.944635e+00  4.939781e+00  4.934196e+00 
##         54353         12752         13427         54364         18029 
##  4.927926e+00  4.927438e+00  4.920837e+00  4.919402e+00  4.916636e+00 
##        103284        117197     100043911         12609         12176 
##  4.913035e+00  4.902852e+00  4.892016e+00  4.891592e+00  4.888191e+00 
##        234413         74277        192652        319924         24136 
##  4.887584e+00  4.886192e+00  4.882725e+00  4.881845e+00  4.880772e+00 
##        217370        321000         74340         72151         16835 
##  4.874231e+00  4.872574e+00  4.869020e+00  4.867178e+00  4.849535e+00 
##         76787         69186         68349         73827         77721 
##  4.847769e+00  4.844056e+00  4.836946e+00  4.836392e+00  4.834232e+00 
##         20375        330216         27388         19212        414801 
##  4.829053e+00  4.825216e+00  4.821092e+00  4.818680e+00  4.817204e+00 
##     100504186        269033        240354        233489         72318 
##  4.814553e+00  4.805948e+00  4.796763e+00  4.794481e+00  4.792121e+00 
##         67145        223753         16337     100039945         54652 
##  4.779737e+00  4.778284e+00  4.775610e+00  4.770724e+00  4.767031e+00 
##         13829         17135         11669         66054     100642166 
##  4.764285e+00  4.759307e+00  4.758137e+00  4.750724e+00  4.745117e+00 
##         69504        231801        333789         12325         72098 
##  4.741269e+00  4.739297e+00  4.724029e+00  4.721450e+00  4.716404e+00 
##         66096        338368        547253        272158        245886 
##  4.714055e+00  4.712002e+00  4.711837e+00  4.703744e+00  4.703690e+00 
##         20652         19356         21354        279029         57028 
##  4.695810e+00  4.688700e+00  4.688675e+00  4.684771e+00  4.683741e+00 
##        231093         11416         66629         16582         71914 
##  4.681551e+00  4.681380e+00  4.677605e+00  4.675662e+00  4.666345e+00 
##        331392         23950        211007     100038504         68646 
##  4.666141e+00  4.649032e+00  4.645434e+00  4.643049e+00  4.641135e+00 
##         13184         75430         76142        211548        100226 
##  4.640342e+00  4.639114e+00  4.633613e+00  4.631263e+00  4.627561e+00 
##     101056044     108168883         18613     102631780        231380 
##  4.623493e+00  4.616918e+00  4.614749e+00  4.614225e+00  4.608486e+00 
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##        109552     100415971        231637         17684         85308 
##  4.548295e+00  4.545802e+00  4.545156e+00  4.544134e+00  4.535756e+00 
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##  4.533933e+00  4.532937e+00  4.526753e+00  4.526018e+00  4.524206e+00 
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##  4.519704e+00  4.517210e+00  4.516433e+00  4.514943e+00  4.507274e+00 
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##  4.506355e+00  4.505853e+00  4.502180e+00  4.492133e+00  4.487891e+00 
##        231866         15184        225994        118445         67661 
##  4.480630e+00  4.478540e+00  4.472092e+00  4.471033e+00  4.470795e+00 
##        106143     100859931        214058        103710         97440 
##  4.465974e+00  4.465885e+00  4.454115e+00  4.452365e+00  4.449305e+00 
##         26401         53895         29806         78246         12192 
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##  4.433254e+00  4.432320e+00  4.420461e+00  4.417128e+00  4.414969e+00 
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##  4.410587e+00  4.406719e+00  4.404722e+00  4.403982e+00  4.399294e+00 
##        237542        234734         18648        225895         99349 
##  4.396182e+00  4.386760e+00  4.377898e+00  4.375818e+00  4.369360e+00 
##         64143        217149        241638        383862         98999 
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##        625631        109674         66706         56541         67229 
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##         20639         78829         68770        664702        216805 
##  4.325205e+00  4.315993e+00  4.314281e+00  4.314030e+00  4.311170e+00 
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##        408068        217356        114584        230577     100504007 
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##         16878        382090        212980         72772         77031 
##  3.693729e+00  3.692213e+00  3.690615e+00  3.685124e+00  3.683450e+00 
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##  3.669572e+00  3.669437e+00  3.668997e+00  3.666883e+00  3.665145e+00 
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##  3.647067e+00  3.645445e+00  3.635363e+00  3.632745e+00  3.631347e+00 
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##  3.582888e+00  3.582004e+00  3.581357e+00  3.574797e+00  3.573587e+00 
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##  3.399757e+00  3.391546e+00  3.391006e+00  3.390821e+00  3.390149e+00 
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##  3.374187e+00  3.363130e+00  3.362013e+00  3.352608e+00  3.350824e+00 
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##  3.347783e+00  3.344056e+00  3.342252e+00  3.333746e+00  3.332591e+00 
##     101055903         22704         74569        104318        238037 
##  3.331846e+00  3.328507e+00  3.327316e+00  3.324107e+00  3.322843e+00 
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##  3.300423e+00  3.297522e+00  3.294914e+00  3.290566e+00  3.289074e+00 
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##  3.286359e+00  3.286315e+00  3.285852e+00  3.282343e+00  3.281643e+00 
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##  3.260696e+00  3.260247e+00  3.254990e+00  3.253701e+00  3.253461e+00 
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##  3.234717e+00  3.233287e+00  3.231544e+00  3.229217e+00  3.227898e+00 
##         18846         64540         56380        228139        382423 
##  3.225525e+00  3.225187e+00  3.220799e+00  3.220359e+00  3.217795e+00 
##         56513         85030        321007         67242         73836 
##  3.217671e+00  3.215554e+00  3.212197e+00  3.208660e+00  3.205324e+00 
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##  3.185393e+00  3.184791e+00  3.179559e+00  3.177072e+00  3.174675e+00 
##         58172         16859        240087         67819         72635 
##  3.169390e+00  3.168687e+00  3.163804e+00  3.161418e+00  3.158123e+00 
##        223267         14388         78733         14682     102638847 
##  3.157148e+00  3.153134e+00  3.151992e+00  3.148168e+00  3.133460e+00 
##        665211        381201        171171         11877        101358 
##  3.126364e+00  3.125390e+00  3.125077e+00  3.122753e+00  3.121304e+00 
##        211666        317757        217069         71801         75560 
##  3.120009e+00  3.119122e+00  3.117858e+00  3.116715e+00  3.108777e+00 
##        331026         22165         12428        620213         76959 
##  3.103832e+00  3.103138e+00  3.100540e+00  3.098498e+00  3.096900e+00 
##        319336         67538        238023        328110         74841 
##  3.091891e+00  3.088884e+00  3.087899e+00  3.086527e+00  3.085558e+00 
##     100039805         67693     102634359        234729        381820 
##  3.085052e+00  3.080766e+00  3.076711e+00  3.076410e+00  3.069114e+00 
##     100038703         70615         99889         78619        224624 
##  3.065250e+00  3.063473e+00  3.054269e+00  3.053693e+00  3.051047e+00 
##        108150         58180        241296         66457         11443 
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##         66071         22282         97423         76367         74052 
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##  3.025847e+00  3.022595e+00  3.022513e+00  3.022219e+00  3.019288e+00 
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##  3.014127e+00  3.013490e+00  3.012806e+00  3.011192e+00  3.011111e+00 
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##  3.005434e+00  3.004774e+00  2.999591e+00  2.999304e+00  2.997731e+00 
##        320148         59008         59016        109154        224023 
##  2.994923e+00  2.992236e+00  2.990672e+00  2.989556e+00  2.989354e+00 
##         68897        207792        231326         23948         69091 
##  2.988067e+00  2.983300e+00  2.982851e+00  2.981370e+00  2.980125e+00 
##         66877         81877        244144         56218         67711 
##  2.979892e+00  2.979343e+00  2.975374e+00  2.974883e+00  2.973543e+00 
##         66251        329358         27406         70266         66125 
##  2.973185e+00  2.972135e+00  2.968050e+00  2.966052e+00  2.964061e+00 
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##  2.963998e+00  2.962326e+00  2.962215e+00  2.960227e+00  2.958508e+00 
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##         68090        432789         66973        546767         67236 
##  2.932833e+00  2.932365e+00  2.929353e+00  2.928830e+00  2.927827e+00 
##        212862        105651        432537         21766     100048701 
##  2.926650e+00  2.923117e+00  2.922431e+00  2.921416e+00  2.919880e+00 
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##  2.919126e+00  2.918125e+00  2.915570e+00  2.913900e+00  2.913099e+00 
##         76282     100503924         72117         20018         76000 
##  2.912877e+00  2.912293e+00  2.912273e+00  2.909853e+00  2.908740e+00 
##         71901         76482         56760        227622         13560 
##  2.907865e+00  2.907612e+00  2.903970e+00  2.903727e+00  2.902642e+00 
##         13170     100417531         69770         66985         56844 
##  2.901161e+00  2.900461e+00  2.898662e+00  2.893117e+00  2.887226e+00 
##        252973         12862        213068         74012         27376 
##  2.886784e+00  2.881997e+00  2.881068e+00  2.878916e+00  2.877953e+00 
##        233079        622568        243373         11858         66421 
##  2.876612e+00  2.875688e+00  2.869723e+00  2.869305e+00  2.869085e+00 
##        231464        245827         14281         68815     100329140 
##  2.866596e+00  2.864156e+00  2.863992e+00  2.861116e+00  2.859049e+00 
##         66164        208144        108707         76987         16848 
##  2.858864e+00  2.857882e+00  2.857783e+00  2.857328e+00  2.854958e+00 
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##  2.851943e+00  2.849545e+00  2.848154e+00  2.847104e+00  2.846373e+00 
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##  2.350360e+00  2.347524e+00  2.345736e+00  2.344335e+00  2.342261e+00 
##        546907     105243585        108100         75415        433456 
##  2.340278e+00  2.340144e+00  2.339234e+00  2.337324e+00  2.336595e+00 
##         14155         76366         77522         19704         20679 
##  2.335743e+00  2.331693e+00  2.331638e+00  2.331205e+00  2.330836e+00 
##        381067     100504130         12759         69745         21415 
##  2.325979e+00  2.322436e+00  2.320551e+00  2.318003e+00  2.315267e+00 
##         71147         17864         69440        208092     100040502 
##  2.314472e+00  2.312183e+00  2.311930e+00  2.311192e+00  2.310515e+00 
##        666473         79554        415115         71131        668887 
##  2.309549e+00  2.308713e+00  2.305350e+00  2.304433e+00  2.303374e+00 
##     105245244         27387        211134         12050         24109 
##  2.299739e+00  2.298943e+00  2.298597e+00  2.294257e+00  2.291735e+00 
##        268465        668677        633498        626848        193813 
##  2.291278e+00  2.291015e+00  2.289827e+00  2.289494e+00  2.287811e+00 
##         19707         14635         26372         18595     100504127 
##  2.286346e+00  2.284659e+00  2.284455e+00  2.283273e+00  2.281508e+00 
##         29810        100072         66290         78834         20443 
##  2.280808e+00  2.279603e+00  2.277224e+00  2.276143e+00  2.274105e+00 
##        270151     100302595         70036         66337     100416455 
##  2.272561e+00  2.270168e+00  2.264359e+00  2.263923e+00  2.263512e+00 
##        105782         54651         52713         16803     115489598 
##  2.261753e+00  2.261021e+00  2.260149e+00  2.259641e+00  2.258043e+00 
##     100417659         69641         56312         66129         67760 
##  2.256447e+00  2.256359e+00  2.253611e+00  2.253181e+00  2.253046e+00 
##        269997         18120        217732         67009         50994 
##  2.251251e+00  2.250808e+00  2.250153e+00  2.248493e+00  2.248459e+00 
##     115488819         66422         73174        230126        233490 
##  2.247820e+00  2.246234e+00  2.246168e+00  2.246046e+00  2.242405e+00 
##         20846        236792         66263         20532        170644 
##  2.241243e+00  2.240894e+00  2.240260e+00  2.238730e+00  2.238664e+00 
##         67675         67101         56486     115487448        207212 
##  2.238549e+00  2.238187e+00  2.238053e+00  2.234678e+00  2.233257e+00 
##        246257     115490433         73467         68087         99470 
##  2.230299e+00  2.229721e+00  2.225092e+00  2.224878e+00  2.224594e+00 
##         56802     102637720         72415         14797         12053 
##  2.222845e+00  2.222761e+00  2.221687e+00  2.216054e+00  2.215216e+00 
##         66523         12122         21899         55942         68192 
##  2.213489e+00  2.212944e+00  2.211860e+00  2.210674e+00  2.205719e+00 
##        270624        225929         69161         76416         78783 
##  2.205464e+00  2.203646e+00  2.199485e+00  2.198279e+00  2.196754e+00 
##         78244         69823        320152         23921         76072 
##  2.195337e+00  2.193642e+00  2.190870e+00  2.190164e+00  2.188941e+00 
##         69004         70031         18141        216618         24105 
##  2.188260e+00  2.186994e+00  2.186722e+00  2.186152e+00  2.185322e+00 
##        625249         21847        319475        631577     125488414 
##  2.184281e+00  2.180775e+00  2.180686e+00  2.180302e+00  2.178696e+00 
##         72205     100038999        320474         70661         60425 
##  2.176742e+00  2.175756e+00  2.175334e+00  2.175259e+00  2.174664e+00 
##         11601        621699         59045         26448         99512 
##  2.170854e+00  2.170522e+00  2.169906e+00  2.169411e+00  2.168772e+00 
##        319545     100217365         78558         66498        213980 
##  2.168708e+00  2.168685e+00  2.168323e+00  2.167131e+00  2.165053e+00 
##         78523         69270         12028        101240         77492 
##  2.163318e+00  2.161475e+00  2.153028e+00  2.152806e+00  2.152227e+00 
##        105727        235043         17259         20597         71913 
##  2.149124e+00  2.145802e+00  2.145459e+00  2.144856e+00  2.144507e+00 
##     102635051        668166        231327        320312         78908 
##  2.144482e+00  2.142657e+00  2.139693e+00  2.137406e+00  2.133944e+00 
##         17128        270163         71832        668045        108673 
##  2.132572e+00  2.132518e+00  2.131447e+00  2.131093e+00  2.129504e+00 
##        208194        214663         66730        214812         78777 
##  2.125816e+00  2.121806e+00  2.121175e+00  2.121119e+00  2.120519e+00 
##        433885        107993         74410         60441     102639315 
##  2.120094e+00  2.116976e+00  2.116002e+00  2.114991e+00  2.113600e+00 
##         30853         12836        270802        381406         69487 
##  2.112018e+00  2.110400e+00  2.109815e+00  2.109268e+00  2.107370e+00 
##         18797         67976        216846        665553         20810 
##  2.106901e+00  2.105490e+00  2.105038e+00  2.103841e+00  2.102322e+00 
##         68481         23955        107971        620253        106795 
##  2.101450e+00  2.099690e+00  2.099220e+00  2.098215e+00  2.097200e+00 
##     115488304         26408         20525         19713        217218 
##  2.096931e+00  2.093078e+00  2.092830e+00  2.092278e+00  2.089901e+00 
##         72655        102926         75284         66525         71101 
##  2.088644e+00  2.088067e+00  2.085629e+00  2.084047e+00  2.083036e+00 
##        102141         14020         70591         71707        226901 
##  2.082657e+00  2.081138e+00  2.079834e+00  2.079816e+00  2.076952e+00 
##         13712         73668     100042498         30840     112136084 
##  2.073008e+00  2.069928e+00  2.069574e+00  2.068751e+00  2.067235e+00 
##        269615         71538         66488         15270         77533 
##  2.066918e+00  2.066746e+00  2.065815e+00  2.064445e+00  2.060171e+00 
##         12617        218850        330941         77381     105244114 
##  2.059108e+00  2.055026e+00  2.054344e+00  2.053142e+00  2.052501e+00 
##         68988         12449         19271         17868         72195 
##  2.051250e+00  2.046886e+00  2.045645e+00  2.044531e+00  2.042910e+00 
##        668300         75692     100327264         12738        114674 
##  2.042012e+00  2.041370e+00  2.039940e+00  2.039682e+00  2.038889e+00 
##        667014        625534         68350        330050         22666 
##  2.038570e+00  2.038138e+00  2.037708e+00  2.032638e+00  2.030095e+00 
##         66943     100137727         70359         98870         14593 
##  2.027987e+00  2.027713e+00  2.027312e+00  2.027084e+00  2.025734e+00 
##         68556         13448         19058        668253         18807 
##  2.025733e+00  2.024142e+00  2.023757e+00  2.023453e+00  2.023090e+00 
##         79044        319653         66513         19899         69008 
##  2.022562e+00  2.022057e+00  2.021658e+00  2.019047e+00  2.018054e+00 
##         78353        209018     102640266         14872         20461 
##  2.017275e+00  2.016513e+00  2.014761e+00  2.014321e+00  2.013610e+00 
##        208691        234663        230967         21678         74614 
##  2.010066e+00  2.009702e+00  2.009367e+00  2.009107e+00  2.008549e+00 
##        100900        230752        634967        230088         15159 
##  2.005778e+00  2.003735e+00  2.002822e+00  2.002766e+00  2.001409e+00 
##         16186         19336        211550        216742        384018 
##  1.993875e+00  1.993157e+00  1.991381e+00  1.990246e+00  1.990182e+00 
##        668453         28077         67939        234595        654803 
##  1.989188e+00  1.985033e+00  1.983423e+00  1.977856e+00  1.977190e+00 
##        320435        237246        270110        252967     102633692 
##  1.976937e+00  1.976877e+00  1.975499e+00  1.974917e+00  1.974383e+00 
##         69478         18676         72145        224116        668563 
##  1.973989e+00  1.972642e+00  1.966687e+00  1.965180e+00  1.962385e+00 
##         30959        433256         14088         66056     100504333 
##  1.962033e+00  1.961498e+00  1.960397e+00  1.956634e+00  1.956318e+00 
##        231863         11540         26417         21956         15201 
##  1.954791e+00  1.954778e+00  1.951028e+00  1.948920e+00  1.947596e+00 
##         18016         73490        170752         50930         12268 
##  1.947005e+00  1.944420e+00  1.944203e+00  1.941661e+00  1.940998e+00 
##        230810         66400         17346         94280         13001 
##  1.939587e+00  1.936527e+00  1.931943e+00  1.931195e+00  1.930860e+00 
##        232975        320362         16151         21991         71900 
##  1.930241e+00  1.929993e+00  1.929566e+00  1.929009e+00  1.928001e+00 
##     100126228         77035         16797        240660         70681 
##  1.926520e+00  1.916339e+00  1.915234e+00  1.913373e+00  1.912146e+00 
##     105246766        242939         20193         12770         74041 
##  1.912034e+00  1.910525e+00  1.909903e+00  1.909862e+00  1.908677e+00 
##        237500        224836        434130         13030        108900 
##  1.907566e+00  1.905524e+00  1.904883e+00  1.904832e+00  1.904419e+00 
##         28105         73067         71804         14312         69752 
##  1.903531e+00  1.903306e+00  1.902844e+00  1.902008e+00  1.898387e+00 
##        110196         51902         66437        381560         17873 
##  1.897713e+00  1.895495e+00  1.889478e+00  1.886359e+00  1.884947e+00 
##         55946     100417389         74316         64009         77559 
##  1.884728e+00  1.881287e+00  1.881094e+00  1.877305e+00  1.877067e+00 
##        102595         55935         20016         51789        104111 
##  1.875212e+00  1.871311e+00  1.869806e+00  1.865774e+00  1.862881e+00 
##         67763         56473         67946         14225         75909 
##  1.860801e+00  1.859300e+00  1.857575e+00  1.857326e+00  1.855244e+00 
##         75668         58217        664947     100532729        321019 
##  1.854039e+00  1.852953e+00  1.851276e+00  1.850903e+00  1.850584e+00 
##         80859         70237         67005         50788         72039 
##  1.850399e+00  1.849196e+00  1.847237e+00  1.844721e+00  1.844704e+00 
##         56771         66935         26426        434782         66658 
##  1.844124e+00  1.841255e+00  1.841146e+00  1.840917e+00  1.839248e+00 
##     100502757         71148        106572        223723         74366 
##  1.839220e+00  1.838891e+00  1.837836e+00  1.837664e+00  1.833260e+00 
##         18109         77402         22032     115489693     102633236 
##  1.831667e+00  1.830436e+00  1.830344e+00  1.830274e+00  1.829469e+00 
##         52575         17274        108907         19727         26458 
##  1.826685e+00  1.825330e+00  1.823899e+00  1.823815e+00  1.822080e+00 
##         77532        213760         56692         12833     101056023 
##  1.821739e+00  1.821537e+00  1.821257e+00  1.821211e+00  1.820380e+00 
##        242691        213121        320204         20354         77779 
##  1.819572e+00  1.811671e+00  1.809934e+00  1.808007e+00  1.807611e+00 
##        381651        240442         22756         76073         66818 
##  1.807380e+00  1.804733e+00  1.804733e+00  1.801644e+00  1.800402e+00 
##         67890         75687         27984        666468     102634576 
##  1.799797e+00  1.799141e+00  1.797529e+00  1.794693e+00  1.792614e+00 
##        237940         21357         20347        102075     100043040 
##  1.791905e+00  1.791246e+00  1.790933e+00  1.790804e+00  1.789625e+00 
##     102639127         67945         54128         18023     100038591 
##  1.788743e+00  1.788043e+00  1.785766e+00  1.785040e+00  1.782375e+00 
##        171429         69780         66610        101602        665367 
##  1.782066e+00  1.782035e+00  1.780245e+00  1.777882e+00  1.774605e+00 
##         67181         54170         12672     100502619         19652 
##  1.774462e+00  1.773172e+00  1.772548e+00  1.772010e+00  1.770879e+00 
##        111116        229644         64933        629170         14580 
##  1.767940e+00  1.765412e+00  1.764004e+00  1.760225e+00  1.758940e+00 
##        269209         56843        320642        212114        238317 
##  1.757933e+00  1.757574e+00  1.756339e+00  1.756168e+00  1.754757e+00 
##         66050         73166        109075         76223         72692 
##  1.752921e+00  1.752558e+00  1.748215e+00  1.747013e+00  1.746411e+00 
##        212547         72361         69537         56299     100503810 
##  1.744672e+00  1.744313e+00  1.743967e+00  1.743311e+00  1.742993e+00 
##         71722         69234        101612         94045         74998 
##  1.742405e+00  1.741764e+00  1.741610e+00  1.741267e+00  1.740908e+00 
##     102634940         75323        110954     108167994        319293 
##  1.740362e+00  1.740022e+00  1.738200e+00  1.734002e+00  1.731895e+00 
##         13542        170789         14547     100416762        269134 
##  1.731780e+00  1.728407e+00  1.728390e+00  1.727671e+00  1.724997e+00 
##     100040633         67851     100042319         66760        114654 
##  1.723553e+00  1.722278e+00  1.721242e+00  1.716816e+00  1.715832e+00 
##         17857        235028         54123        110750         84653 
##  1.715386e+00  1.715385e+00  1.713427e+00  1.712555e+00  1.711257e+00 
##         76233         19157         71711         29808        231872 
##  1.710681e+00  1.708181e+00  1.708075e+00  1.707744e+00  1.707731e+00 
##         69820     102638940         74440        235509         16716 
##  1.706746e+00  1.702269e+00  1.701428e+00  1.698333e+00  1.696057e+00 
##         13163         22712        545539     100043431         22388 
##  1.695728e+00  1.694033e+00  1.693953e+00  1.692512e+00  1.691892e+00 
##         28146         26356         13004        320376        386506 
##  1.691676e+00  1.691655e+00  1.690171e+00  1.689960e+00  1.689218e+00 
##         14163         83602        547166         56018         13731 
##  1.688484e+00  1.686905e+00  1.686551e+00  1.685147e+00  1.684304e+00 
##        624251        107250        319236        103963         19143 
##  1.683826e+00  1.683771e+00  1.683744e+00  1.682624e+00  1.680728e+00 
##     102640742        230809        243912        230751         18181 
##  1.679465e+00  1.676697e+00  1.676355e+00  1.673525e+00  1.672913e+00 
##         67657        319887        232878        102436         76612 
##  1.668448e+00  1.666729e+00  1.665369e+00  1.664665e+00  1.663803e+00 
##         27399        231668        114332         64082         74584 
##  1.663370e+00  1.661141e+00  1.661030e+00  1.660272e+00  1.660142e+00 
##         69802        264134         15932         19152         18640 
##  1.659926e+00  1.659478e+00  1.658921e+00  1.656808e+00  1.655333e+00 
##         12868         67158         11567         54411         54189 
##  1.654463e+00  1.653812e+00  1.653626e+00  1.652785e+00  1.652343e+00 
##         17245        433182        230641        625660         80281 
##  1.651058e+00  1.649571e+00  1.649234e+00  1.645380e+00  1.644979e+00 
##        231549        243983        230903        269423     102466214 
##  1.644578e+00  1.644137e+00  1.643604e+00  1.641808e+00  1.640114e+00 
##         75995        170823        399510         13555         12455 
##  1.639021e+00  1.634169e+00  1.633121e+00  1.629935e+00  1.629418e+00 
##         93734         83396         13434     102641476        107197 
##  1.629098e+00  1.628122e+00  1.627110e+00  1.626645e+00  1.625896e+00 
##         17686         14729         66445         78512        674228 
##  1.625270e+00  1.624461e+00  1.624438e+00  1.624038e+00  1.622639e+00 
##         67914         54152        320056        621446     100310753 
##  1.622409e+00  1.622316e+00  1.622060e+00  1.621504e+00  1.620302e+00 
##        402737         20533         69544         23863        102060 
##  1.618510e+00  1.616898e+00  1.616538e+00  1.613079e+00  1.610637e+00 
##        116748         79233        234396     100040190         14969 
##  1.607623e+00  1.607493e+00  1.607147e+00  1.606654e+00  1.606342e+00 
##        217310        381258        227541        625989         68737 
##  1.605923e+00  1.605198e+00  1.605126e+00  1.604844e+00  1.602407e+00 
##        218734         20610         68153        434148     100038526 
##  1.601152e+00  1.601076e+00  1.600439e+00  1.598927e+00  1.597153e+00 
##         69548        209012         14256         74766        210766 
##  1.596772e+00  1.596244e+00  1.595285e+00  1.594275e+00  1.592817e+00 
##         64295        621763         27278         69129     102636299 
##  1.592430e+00  1.592250e+00  1.591364e+00  1.590031e+00  1.589615e+00 
##     100040608     102634324         75870         22022         12262 
##  1.589269e+00  1.588397e+00  1.586037e+00  1.585667e+00  1.585642e+00 
##         69740     108168031         66676         71856        667745 
##  1.583684e+00  1.583070e+00  1.582221e+00  1.580527e+00  1.580335e+00 
##         73172         78408        232933        629471         69191 
##  1.579416e+00  1.579047e+00  1.575933e+00  1.575560e+00  1.575404e+00 
##        101985         67784        320162        547431        320757 
##  1.575031e+00  1.574419e+00  1.574239e+00  1.573899e+00  1.572381e+00 
##        320100     102465604         15412         75646        230257 
##  1.572316e+00  1.571830e+00  1.570450e+00  1.570131e+00  1.569884e+00 
##        320438         14840        217353        320685         11993 
##  1.566640e+00  1.566509e+00  1.565519e+00  1.564841e+00  1.563578e+00 
##     102632739         67349     100504312        109267         56438 
##  1.558709e+00  1.558421e+00  1.557051e+00  1.555345e+00  1.554891e+00 
##         17827        381921         14772         17350        635136 
##  1.554200e+00  1.553521e+00  1.552557e+00  1.552322e+00  1.550885e+00 
##         53599         69694         73204         68281        229524 
##  1.550355e+00  1.550032e+00  1.549628e+00  1.549570e+00  1.549482e+00 
##         71968         76166         74522         22693         70207 
##  1.548747e+00  1.547402e+00  1.546199e+00  1.545567e+00  1.543710e+00 
##         69168     102632031        242557         66521         70458 
##  1.542725e+00  1.540363e+00  1.539952e+00  1.539437e+00  1.538705e+00 
##         12841         26456         56703         81630        620269 
##  1.536037e+00  1.536013e+00  1.535671e+00  1.532071e+00  1.531558e+00 
##         66308         54199         54216        382510         17068 
##  1.528377e+00  1.528361e+00  1.527940e+00  1.526025e+00  1.525740e+00 
##         73205     100039814        109901     100040769         70957 
##  1.524335e+00  1.522777e+00  1.522650e+00  1.521905e+00  1.521521e+00 
##         74018         71169        228543         57330         13616 
##  1.521461e+00  1.520782e+00  1.519650e+00  1.519608e+00  1.519516e+00 
##         14841         18634         68927         13821        224481 
##  1.518721e+00  1.516372e+00  1.514908e+00  1.512639e+00  1.512601e+00 
##         70351         66975        106073         67893         13392 
##  1.512403e+00  1.511045e+00  1.510149e+00  1.509440e+00  1.508901e+00 
##     102636118        235293     100038388        218214     102640165 
##  1.506754e+00  1.506539e+00  1.504647e+00  1.503785e+00  1.503175e+00 
##        382114         74487        245671        319162        667700 
##  1.502053e+00  1.500408e+00  1.500048e+00  1.498879e+00  1.495929e+00 
##        232533         70345         19943     102638630     100503915 
##  1.495457e+00  1.495359e+00  1.495319e+00  1.495128e+00  1.492514e+00 
##        233912         57816     102636679         67513         74114 
##  1.490743e+00  1.489806e+00  1.489200e+00  1.489157e+00  1.487722e+00 
##     105244010         67130         19824         69085     100040505 
##  1.487023e+00  1.486745e+00  1.486061e+00  1.484825e+00  1.484665e+00 
##        230587        667410         66431         15202        192662 
##  1.483921e+00  1.480773e+00  1.480578e+00  1.480573e+00  1.480315e+00 
##        236794         56460         72147        105000         14884 
##  1.479596e+00  1.479053e+00  1.478227e+00  1.476815e+00  1.475788e+00 
##        170787        109660        232934         84682         69572 
##  1.475670e+00  1.475069e+00  1.473710e+00  1.470092e+00  1.469989e+00 
##         72843        230936        231329     105246071        620915 
##  1.468829e+00  1.468322e+00  1.467512e+00  1.466331e+00  1.464936e+00 
##         67898         69773        414123        171486        216274 
##  1.463951e+00  1.462804e+00  1.462742e+00  1.456382e+00  1.454965e+00 
##        209815         56190        235587         77446        668245 
##  1.454337e+00  1.453362e+00  1.452612e+00  1.452458e+00  1.450746e+00 
##         78521         68521     100862302     100039501     105244354 
##  1.449609e+00  1.447240e+00  1.446434e+00  1.446301e+00  1.440415e+00 
##         66443         71275         66315     102638353         77430 
##  1.440328e+00  1.440215e+00  1.439350e+00  1.437779e+00  1.436716e+00 
##         66925         66489        212398         13559         71876 
##  1.436144e+00  1.433927e+00  1.433838e+00  1.432643e+00  1.430958e+00 
##     100503659         71004         16816         66067         12763 
##  1.428823e+00  1.428717e+00  1.427641e+00  1.426037e+00  1.424526e+00 
##         18130        240549        104383        100604         67263 
##  1.422618e+00  1.422592e+00  1.422577e+00  1.421463e+00  1.420742e+00 
##        140488         52609         64059         26949        103534 
##  1.420621e+00  1.419717e+00  1.417671e+00  1.417646e+00  1.416339e+00 
##         16562        212198        241274     100306959        442802 
##  1.413797e+00  1.412714e+00  1.412506e+00  1.411810e+00  1.411733e+00 
##        326622        210573        230119         12775         74670 
##  1.410015e+00  1.407076e+00  1.406819e+00  1.406696e+00  1.406222e+00 
##        114565     100039027         74559        665751         70380 
##  1.404205e+00  1.403853e+00  1.401875e+00  1.401697e+00  1.401468e+00 
##         72778         20401         67943         19060     105246363 
##  1.401456e+00  1.400593e+00  1.400301e+00  1.400135e+00  1.399810e+00 
##        109910        668173         20320        621146     100169878 
##  1.398413e+00  1.398262e+00  1.398126e+00  1.397191e+00  1.396420e+00 
##        321008         67605         67369        664830         69136 
##  1.395175e+00  1.394955e+00  1.394677e+00  1.393663e+00  1.393102e+00 
##        628412         72055     100041399        231887         66971 
##  1.392449e+00  1.391894e+00  1.390864e+00  1.390025e+00  1.389642e+00 
##         22172         68166         20469        242838        637776 
##  1.387809e+00  1.386856e+00  1.385878e+00  1.385677e+00  1.384971e+00 
##        319848        606735        230674        330902        231630 
##  1.382670e+00  1.380992e+00  1.379000e+00  1.378289e+00  1.377679e+00 
##        216081         66595        117591     100504201         76311 
##  1.376969e+00  1.376691e+00  1.376515e+00  1.376488e+00  1.376486e+00 
##         74754        404381         14865         66840        629820 
##  1.374412e+00  1.371699e+00  1.368905e+00  1.368528e+00  1.368418e+00 
##     100042465        625424        544922     100124623         22215 
##  1.365202e+00  1.361817e+00  1.359758e+00  1.359490e+00  1.359190e+00 
##        228983        620782         11468        233115        242584 
##  1.357984e+00  1.357661e+00  1.356105e+00  1.354998e+00  1.354717e+00 
##         81003        276809        545975         72822     100859962 
##  1.354694e+00  1.353175e+00  1.353108e+00  1.352983e+00  1.352539e+00 
##         18793        380608        101502        228785         70617 
##  1.352338e+00  1.352190e+00  1.352104e+00  1.348862e+00  1.347645e+00 
##         66593         72614        236790         16580        671439 
##  1.347094e+00  1.346681e+00  1.346491e+00  1.345926e+00  1.344659e+00 
##         20662        320159         12151        216019         13650 
##  1.344560e+00  1.344373e+00  1.341839e+00  1.340575e+00  1.340259e+00 
##        114255        380614     102637672     100504586        213233 
##  1.337197e+00  1.336850e+00  1.336426e+00  1.333625e+00  1.333569e+00 
##         68133     105242826         26894     100504360         24075 
##  1.331925e+00  1.330836e+00  1.328874e+00  1.328033e+00  1.327645e+00 
##        216157         16653        667572         56314     100417950 
##  1.325369e+00  1.323871e+00  1.323068e+00  1.323025e+00  1.322451e+00 
##        217869         17687         16211     100038429         27015 
##  1.321546e+00  1.319920e+00  1.319359e+00  1.319310e+00  1.317410e+00 
##         68126         20482        652920         12856        109093 
##  1.313160e+00  1.312523e+00  1.311275e+00  1.310545e+00  1.308140e+00 
##         11984        116848         52432         75690         67170 
##  1.307483e+00  1.307113e+00  1.306261e+00  1.304142e+00  1.302717e+00 
##         73804     100043387     100038474        234479        110542 
##  1.302226e+00  1.301963e+00  1.301553e+00  1.301531e+00  1.301368e+00 
##         67978        194388         13619     102640440         70435 
##  1.301148e+00  1.300923e+00  1.300818e+00  1.300574e+00  1.300319e+00 
##     115488998         73182        242418         77219     100503381 
##  1.299981e+00  1.297591e+00  1.297296e+00  1.295481e+00  1.295053e+00 
##         13498         26901        214106        171194         67418 
##  1.294104e+00  1.293633e+00  1.293198e+00  1.292079e+00  1.291033e+00 
##        170758         72672        102920        628239         22177 
##  1.290705e+00  1.290202e+00  1.290021e+00  1.289990e+00  1.289417e+00 
##         67885         14586         73711         20355         13340 
##  1.287259e+00  1.286263e+00  1.286171e+00  1.285094e+00  1.284856e+00 
##        240186         78618         52397        621710        213053 
##  1.282903e+00  1.282634e+00  1.280238e+00  1.280219e+00  1.279027e+00 
##         75617         22385         84092        378462         71639 
##  1.278901e+00  1.278545e+00  1.277771e+00  1.277401e+00  1.276148e+00 
##         76789         22224        622320     100038629         73251 
##  1.275964e+00  1.274352e+00  1.273325e+00  1.271847e+00  1.271598e+00 
##         59049         74198         67252         53418         76800 
##  1.270833e+00  1.270807e+00  1.270629e+00  1.270412e+00  1.270015e+00 
##         71738        667370     115488907         52118        544719 
##  1.269459e+00  1.269420e+00  1.269011e+00  1.268680e+00  1.267788e+00 
##        225467        272589        381350        627717     101055953 
##  1.266787e+00  1.266678e+00  1.265400e+00  1.264149e+00  1.263364e+00 
##        382406         53378        170772        110006     100040344 
##  1.262341e+00  1.261194e+00  1.259682e+00  1.259544e+00  1.259172e+00 
##        330921         19156         16782        403202         76737 
##  1.256968e+00  1.256185e+00  1.255912e+00  1.255550e+00  1.255098e+00 
##        668595     108167416         22035        215446        329470 
##  1.254338e+00  1.254054e+00  1.253382e+00  1.252877e+00  1.252876e+00 
##        625018         22717         75826         66757        319998 
##  1.252842e+00  1.252013e+00  1.251232e+00  1.250968e+00  1.250795e+00 
##     100038566         22123         11833         76547     100040287 
##  1.250118e+00  1.250033e+00  1.250027e+00  1.243788e+00  1.243563e+00 
##        212670        114874     102632467         72313     100043880 
##  1.243533e+00  1.242661e+00  1.242639e+00  1.241849e+00  1.241404e+00 
##         71295         56772         68667         75387         64296 
##  1.240033e+00  1.239944e+00  1.239064e+00  1.237395e+00  1.237283e+00 
##        103466         74846         69151        381199         20510 
##  1.237036e+00  1.236223e+00  1.235933e+00  1.235815e+00  1.235349e+00 
##        433432         71303        108154         77059        107702 
##  1.234383e+00  1.233528e+00  1.233038e+00  1.232263e+00  1.230199e+00 
##        623872         64654        224897         18823         13436 
##  1.230045e+00  1.228968e+00  1.228354e+00  1.227452e+00  1.227400e+00 
##        100986        104570         69918        103268         56857 
##  1.227249e+00  1.227064e+00  1.226722e+00  1.226716e+00  1.226546e+00 
##     115488154         17119        229011         66419         73218 
##  1.225765e+00  1.224469e+00  1.224225e+00  1.220382e+00  1.218236e+00 
##         15126         72973        109284         78329         81897 
##  1.217774e+00  1.217537e+00  1.216871e+00  1.216813e+00  1.216477e+00 
##        545486         67179         51797         14172         68472 
##  1.214498e+00  1.213650e+00  1.213083e+00  1.212863e+00  1.212654e+00 
##         94180         14960        668565         98845        268697 
##  1.211189e+00  1.210668e+00  1.210434e+00  1.210154e+00  1.209767e+00 
##         70080        108037        329575        353258        105732 
##  1.207659e+00  1.207191e+00  1.207041e+00  1.206788e+00  1.206082e+00 
##        667786        232089        237400     100041979         14584 
##  1.205864e+00  1.204453e+00  1.203746e+00  1.203570e+00  1.202502e+00 
##        271849         15958     102638450     100043236         17096 
##  1.202187e+00  1.198989e+00  1.198719e+00  1.197918e+00  1.197666e+00 
##         52468        102339     100503670         18220         66817 
##  1.196734e+00  1.195869e+00  1.194551e+00  1.193753e+00  1.193202e+00 
##         59004        171508     115482680         72999         74013 
##  1.191429e+00  1.190923e+00  1.190072e+00  1.189437e+00  1.189297e+00 
##        654818        104732        231069         77305         56458 
##  1.188628e+00  1.187485e+00  1.187253e+00  1.187247e+00  1.184442e+00 
##         11694         66276     102635760        244373        432767 
##  1.182796e+00  1.182405e+00  1.180699e+00  1.180543e+00  1.179963e+00 
##         68682         26410         26382         77744        433806 
##  1.179839e+00  1.179243e+00  1.178199e+00  1.176898e+00  1.176763e+00 
##         22371         26961        231004         66674         65099 
##  1.176179e+00  1.175575e+00  1.173767e+00  1.173719e+00  1.172396e+00 
##        216169         78610         11430         20964         26370 
##  1.172247e+00  1.171987e+00  1.171853e+00  1.170270e+00  1.169149e+00 
##         12659         27397         13611         30934     100504231 
##  1.168470e+00  1.168316e+00  1.167712e+00  1.167336e+00  1.166363e+00 
##         12873        665478        319370         69859         16210 
##  1.166002e+00  1.165017e+00  1.164730e+00  1.163710e+00  1.163106e+00 
##        240120         54402     100126033     100043806         84035 
##  1.161381e+00  1.160650e+00  1.160118e+00  1.159739e+00  1.159614e+00 
##        214917         69019        667233     102634450         66061 
##  1.159377e+00  1.159159e+00  1.157386e+00  1.156906e+00  1.155802e+00 
##        217835     102639741         18607         67563         11605 
##  1.152494e+00  1.151895e+00  1.151874e+00  1.151449e+00  1.150699e+00 
##        240638     100039709         18604         18294     110599566 
##  1.150481e+00  1.148572e+00  1.148419e+00  1.146568e+00  1.145171e+00 
##         68050        329584        108086        666869         20822 
##  1.144666e+00  1.144387e+00  1.143205e+00  1.142457e+00  1.142214e+00 
##        217169        242646        639787         67277         13360 
##  1.141112e+00  1.138004e+00  1.136069e+00  1.133387e+00  1.133207e+00 
##        108689         74721     100042791     102637874         76781 
##  1.132363e+00  1.131106e+00  1.130767e+00  1.130688e+00  1.130240e+00 
##         52570         69106        635651         74420         54369 
##  1.129786e+00  1.129506e+00  1.129370e+00  1.129189e+00  1.129001e+00 
##        210741         22121        192663         23988        320046 
##  1.128341e+00  1.128269e+00  1.125515e+00  1.124807e+00  1.124521e+00 
##         11740        217463        224598         66477        280668 
##  1.124248e+00  1.123973e+00  1.123450e+00  1.121543e+00  1.120423e+00 
##         68559        104349         13629         68845         66690 
##  1.120028e+00  1.119701e+00  1.119412e+00  1.118769e+00  1.117913e+00 
##         20779     100418227     100418004        223669        242409 
##  1.117778e+00  1.117265e+00  1.116820e+00  1.112739e+00  1.112496e+00 
##         77983         68910         11689     102636239        209448 
##  1.111497e+00  1.109825e+00  1.109473e+00  1.109312e+00  1.109118e+00 
##         68107        329877        414085         66913        193670 
##  1.107234e+00  1.107155e+00  1.101992e+00  1.101639e+00  1.101583e+00 
##        627455         68818        666883         69657     100041823 
##  1.101565e+00  1.101084e+00  1.100691e+00  1.100681e+00  1.099152e+00 
##     115487967         22192        664893        233651         56404 
##  1.099061e+00  1.098843e+00  1.096919e+00  1.095749e+00  1.095239e+00 
##        104401         13400        235416         16780         93687 
##  1.093402e+00  1.091943e+00  1.091885e+00  1.091673e+00  1.090946e+00 
##         66379         14670         54473         67900     100041907 
##  1.090723e+00  1.089875e+00  1.088713e+00  1.088185e+00  1.087702e+00 
##         20102         73713        102115         67674         13497 
##  1.087524e+00  1.087493e+00  1.086743e+00  1.085343e+00  1.084102e+00 
##        667483         19698         18018         14070        226548 
##  1.083944e+00  1.083032e+00  1.082804e+00  1.081662e+00  1.081420e+00 
##         72807        667832         72012        434234         67166 
##  1.080498e+00  1.080304e+00  1.079639e+00  1.079585e+00  1.078451e+00 
##        114642        212281         72180         15894        107373 
##  1.077715e+00  1.076564e+00  1.076114e+00  1.074599e+00  1.072433e+00 
##         74096        231470        117146         74577         68040 
##  1.072316e+00  1.070847e+00  1.070456e+00  1.070101e+00  1.068800e+00 
##         23999         66439        666553         66116     100503374 
##  1.067003e+00  1.065459e+00  1.064316e+00  1.063517e+00  1.061370e+00 
##        627828        194401        100342         20361         18117 
##  1.061001e+00  1.060815e+00  1.060614e+00  1.059922e+00  1.059185e+00 
##        229521        118453         12397         71238         66680 
##  1.058697e+00  1.058566e+00  1.058390e+00  1.058263e+00  1.058241e+00 
##     100502985        667073     100043257        211286     105247253 
##  1.057179e+00  1.057105e+00  1.056666e+00  1.056281e+00  1.055651e+00 
##        320700         69893         26940         71779         66511 
##  1.055631e+00  1.054741e+00  1.054492e+00  1.052595e+00  1.052487e+00 
##        244548        211228        545567        654804         63872 
##  1.052336e+00  1.049780e+00  1.049674e+00  1.049464e+00  1.048959e+00 
##         28006         11551         18587         17991        114714 
##  1.048937e+00  1.048899e+00  1.048211e+00  1.047845e+00  1.047172e+00 
##         72552         22709         75420        666712         67610 
##  1.047075e+00  1.046773e+00  1.044318e+00  1.043927e+00  1.043903e+00 
##         50498        215095        102502         75588        260301 
##  1.043106e+00  1.042194e+00  1.041996e+00  1.040768e+00  1.040707e+00 
##         13806         76813     101055907        619909         12778 
##  1.039887e+00  1.039577e+00  1.038098e+00  1.037370e+00  1.037026e+00 
##     102631889         72575         19726         78339         12070 
##  1.035215e+00  1.035009e+00  1.034126e+00  1.034040e+00  1.033652e+00 
##     102638408         68255     100042107         13666         70375 
##  1.031795e+00  1.031394e+00  1.030115e+00  1.029988e+00  1.029744e+00 
##         50877     100302301        109212        103850         68767 
##  1.025657e+00  1.024718e+00  1.024588e+00  1.023876e+00  1.022935e+00 
##        279572         12715         14114         74392        225898 
##  1.022374e+00  1.020808e+00  1.020262e+00  1.019057e+00  1.018722e+00 
##         54390         67065         78688         73229     100503468 
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##         16572         57783         66058     100416965     102465959 
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##  6.467246e-01  6.466316e-01  6.463198e-01  6.463064e-01  6.462187e-01 
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##  6.380445e-01  6.379426e-01  6.375963e-01  6.373274e-01  6.372322e-01 
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##     100039138     100042734         13063     105886298         67067 
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##        544696        664907        668022        665145     100416968 
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##         20850         93675     100039417     105246016     100503545 
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##        320799         58223         56535     115490136         52705 
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##        667486     115490363        620542         68428     100041925 
##  3.157421e-01  3.150791e-01  3.148767e-01  3.145048e-01  3.144009e-01 
##         15372        270091         83673         13356         71599 
##  3.143533e-01  3.143171e-01  3.142767e-01  3.141378e-01  3.138664e-01 
##         70026        434197        208154         15976         54218 
##  3.137802e-01  3.136708e-01  3.136567e-01  3.135973e-01  3.133145e-01 
##        353327        666802         66496     101669761        330414 
##  3.132966e-01  3.132542e-01  3.129639e-01  3.127840e-01  3.126202e-01 
##         67307        639030         12055     100038392        666809 
##  3.123785e-01  3.118508e-01  3.117699e-01  3.116833e-01  3.110577e-01 
##     115489635         66270        675985     100418393         56443 
##  3.107869e-01  3.106994e-01  3.106176e-01  3.105177e-01  3.104710e-01 
##        665053         16182     100042650         58248         17940 
##  3.104290e-01  3.101066e-01  3.100753e-01  3.098331e-01  3.097617e-01 
##        668829        218460        100683         27205         94315 
##  3.096848e-01  3.093687e-01  3.092591e-01  3.083519e-01  3.082461e-01 
##     100040563     100504389        435329         68510         74039 
##  3.082405e-01  3.080066e-01  3.076445e-01  3.075773e-01  3.075687e-01 
##         19428        623781         17691        242235         19732 
##  3.074390e-01  3.070101e-01  3.067916e-01  3.066327e-01  3.066222e-01 
##     100417819     100504121         67048        442829         68219 
##  3.061066e-01  3.060480e-01  3.060414e-01  3.058866e-01  3.057147e-01 
##         68067        666668     100158261         20516         13972 
##  3.051570e-01  3.048889e-01  3.048611e-01  3.046137e-01  3.032903e-01 
##        102124        433716        385523        319876        664981 
##  3.023269e-01  3.022865e-01  3.017249e-01  3.015831e-01  3.015080e-01 
##        791413         20382     100042925        665129     102902673 
##  3.013146e-01  3.007793e-01  3.004397e-01  3.004025e-01  3.001115e-01 
##        665113         20918         66152     118567529        668845 
##  2.998209e-01  2.992325e-01  2.991831e-01  2.990944e-01  2.990775e-01 
##        622271        110350         55938         70373         67283 
##  2.988790e-01  2.986854e-01  2.984833e-01  2.981591e-01  2.980833e-01 
##        674396        791280         56525         17997     102392594 
##  2.976832e-01  2.974429e-01  2.973733e-01  2.972328e-01  2.971640e-01 
##         52055         76509         17179         66965         17156 
##  2.969233e-01  2.958154e-01  2.954412e-01  2.954114e-01  2.947244e-01 
##         11666         66552        606534         68531        625349 
##  2.946735e-01  2.943297e-01  2.941060e-01  2.940386e-01  2.940049e-01 
##         74969        107986         74554        668991        667891 
##  2.936783e-01  2.934720e-01  2.931167e-01  2.930843e-01  2.926357e-01 
##         17966         69010         21955     100040151        667638 
##  2.926169e-01  2.924574e-01  2.918778e-01  2.916476e-01  2.914564e-01 
##         16828         14630        667558         72946         78286 
##  2.912929e-01  2.909590e-01  2.903847e-01  2.903312e-01  2.903039e-01 
##         74287     100384873     100504390        629709         68794 
##  2.901965e-01  2.897335e-01  2.897079e-01  2.886446e-01  2.882180e-01 
##         17283     115489514     100286840        212989        619324 
##  2.873963e-01  2.869626e-01  2.860474e-01  2.857026e-01  2.856430e-01 
##     115490044        383075         57745        270148         27405 
##  2.853134e-01  2.852092e-01  2.847745e-01  2.841670e-01  2.841163e-01 
##         20907         67778        170719         70928        382985 
##  2.835802e-01  2.830271e-01  2.828106e-01  2.827810e-01  2.827241e-01 
##        623031        668578         21944         75182     108168275 
##  2.823624e-01  2.818439e-01  2.818280e-01  2.817356e-01  2.816671e-01 
##        667947        266692     100306874        382030         20969 
##  2.814440e-01  2.814253e-01  2.809433e-01  2.807096e-01  2.806381e-01 
##        666597        100340     115486186     100534357         76976 
##  2.803407e-01  2.800219e-01  2.797723e-01  2.797002e-01  2.796790e-01 
##         63959         14085        435657         24084     115485611 
##  2.796650e-01  2.794735e-01  2.794172e-01  2.792725e-01  2.792142e-01 
##         66611     100038371         78617     100042226        242474 
##  2.787036e-01  2.781704e-01  2.781487e-01  2.779385e-01  2.778383e-01 
##         13168         18655        213498         30954         72285 
##  2.774476e-01  2.768198e-01  2.766296e-01  2.764232e-01  2.764198e-01 
##         72265     115490291         13082     100044855        106763 
##  2.762635e-01  2.762107e-01  2.762085e-01  2.762025e-01  2.760113e-01 
##         84094        272027         26568     100039489     100049161 
##  2.759967e-01  2.759714e-01  2.754926e-01  2.753271e-01  2.751880e-01 
##         66201         19172        433712         94221         12012 
##  2.745773e-01  2.744573e-01  2.742518e-01  2.741595e-01  2.734096e-01 
##         30843     100041570        170829     102636013     108167954 
##  2.730903e-01  2.728361e-01  2.724528e-01  2.723034e-01  2.720779e-01 
##         17256        320650     100113364         67279         18432 
##  2.718494e-01  2.718255e-01  2.717782e-01  2.717114e-01  2.714192e-01 
##        667937        240614        664920        360213        225638 
##  2.713563e-01  2.711972e-01  2.704884e-01  2.704112e-01  2.703244e-01 
##     100125929     102631840         15040         20615         20405 
##  2.696988e-01  2.694898e-01  2.693865e-01  2.691490e-01  2.690978e-01 
##        629081        108902         67474         20973     100134990 
##  2.690499e-01  2.685742e-01  2.680308e-01  2.677006e-01  2.668255e-01 
##         85031         78214        238333        545790     100417846 
##  2.668197e-01  2.667221e-01  2.666310e-01  2.666099e-01  2.663862e-01 
##         74718         21355         56495        332713         76308 
##  2.663114e-01  2.660940e-01  2.659963e-01  2.659709e-01  2.659413e-01 
##         12359         75616        666456        666143         67489 
##  2.658260e-01  2.656053e-01  2.652923e-01  2.652006e-01  2.651379e-01 
##         74414        245884         66373        319743        192653 
##  2.651246e-01  2.650742e-01  2.650641e-01  2.650393e-01  2.650112e-01 
##         72480         16176         71687         53857        620290 
##  2.649870e-01  2.648174e-01  2.646527e-01  2.644976e-01  2.641174e-01 
##     115489155         20341         73233     105245868        209488 
##  2.637400e-01  2.632669e-01  2.632369e-01  2.629178e-01  2.626011e-01 
##        665380        544884        100383         83984        233038 
##  2.625707e-01  2.621919e-01  2.621586e-01  2.620847e-01  2.620514e-01 
##         17069     100041511        237926         14777        666616 
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##         66491        433968     100039419        624863     100504140 
##  2.597052e-01  2.595590e-01  2.594845e-01  2.594445e-01  2.593406e-01 
##     100379259         15193        383570         15248     100045778 
##  2.592938e-01  2.592783e-01  2.590083e-01  2.585888e-01  2.581213e-01 
##        215819         64424        666514         68969        623208 
##  2.578855e-01  2.578596e-01  2.578265e-01  2.578207e-01  2.566661e-01 
##        668709         15312         98404        231207        666038 
##  2.561538e-01  2.560205e-01  2.556061e-01  2.555842e-01  2.552105e-01 
##         67125         57746         69274         67011        622432 
##  2.551555e-01  2.548028e-01  2.546030e-01  2.544958e-01  2.544621e-01 
##        140765        436100         20678        665250         70460 
##  2.540614e-01  2.536808e-01  2.536405e-01  2.535859e-01  2.534594e-01 
##         71233     100039760        217779     100416680     102465884 
##  2.533039e-01  2.532015e-01  2.529481e-01  2.524400e-01  2.523922e-01 
##        225362     100039302        667903        209753         69089 
##  2.523376e-01  2.521370e-01  2.514433e-01  2.510284e-01  2.509232e-01 
##         16763         56367        108812         76779        268300 
##  2.507247e-01  2.504938e-01  2.504484e-01  2.501792e-01  2.498571e-01 
##        433748        108934        666504         74706     105245369 
##  2.495705e-01  2.492723e-01  2.492480e-01  2.490774e-01  2.488818e-01 
##     100042367        209497        268988         66816         21745 
##  2.488088e-01  2.487852e-01  2.486534e-01  2.482326e-01  2.482006e-01 
##         67746         77980        246228        621815        231296 
##  2.480718e-01  2.479146e-01  2.474061e-01  2.472165e-01  2.471075e-01 
##        666326         67049     100303737         22187        327824 
##  2.467773e-01  2.467123e-01  2.463467e-01  2.463463e-01  2.462189e-01 
##         69876     105244012     115490185     100503307     100038753 
##  2.459757e-01  2.459390e-01  2.457217e-01  2.452090e-01  2.449897e-01 
##        432881     100418033         75964     115489459        545429 
##  2.449891e-01  2.449762e-01  2.448516e-01  2.447775e-01  2.446377e-01 
##         66508         14626        380713        328166        330503 
##  2.446371e-01  2.444922e-01  2.443887e-01  2.443289e-01  2.439583e-01 
##        433230        544800        545459         50931        170729 
##  2.437312e-01  2.436211e-01  2.434064e-01  2.432889e-01  2.430696e-01 
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##  2.430562e-01  2.426150e-01  2.425076e-01  2.423813e-01  2.422908e-01 
##         11844         27049        640094        105835        665828 
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##        665247         30049     100041342        216344     100043832 
##  2.409698e-01  2.409096e-01  2.404054e-01  2.403636e-01  2.402548e-01 
##         56437        545234        666648         67533        242109 
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##  2.384187e-01  2.380492e-01  2.377149e-01  2.374408e-01  2.371991e-01 
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##        321010         12890        631097        619917     108168897 
##  2.363763e-01  2.359049e-01  2.355496e-01  2.354116e-01  2.349817e-01 
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##  2.309829e-01  2.309564e-01  2.305400e-01  2.303325e-01  2.302625e-01 
##        381077         71520        218885     100039988         56088 
##  2.298601e-01  2.295777e-01  2.295041e-01  2.293872e-01  2.292911e-01 
##         19339        668444         69227        258438        545864 
##  2.290075e-01  2.284933e-01  2.284509e-01  2.281392e-01  2.279522e-01 
##     100043205        193736        432508        668955         70425 
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##  2.267982e-01  2.267939e-01  2.266706e-01  2.263706e-01  2.263384e-01 
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##     102466781        668668         56444         97484        320183 
##  2.150438e-01  2.147437e-01  2.141850e-01  2.140213e-01  2.140117e-01 
##        767812        380629        209003         67226        433700 
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##  1.866943e-01  1.865380e-01  1.864274e-01  1.862651e-01  1.861134e-01 
##         16369         17165     115488251     102640532        384484 
##  1.857376e-01  1.856084e-01  1.855521e-01  1.854988e-01  1.854964e-01 
##         20975         73449         97961        239114         20310 
##  1.849071e-01  1.846946e-01  1.845070e-01  1.842281e-01  1.842050e-01 
##        233276        442813     100504608     105247048         68014 
##  1.840309e-01  1.834295e-01  1.833945e-01  1.833933e-01  1.832538e-01 
##        780960         21924         68738        665461     100042165 
##  1.831686e-01  1.829638e-01  1.825929e-01  1.824522e-01  1.823033e-01 
##        235283        100090        328489        213011     100504446 
##  1.820828e-01  1.819829e-01  1.819727e-01  1.817122e-01  1.815907e-01 
##     102636367         56702        667644        271005         78312 
##  1.811938e-01  1.808828e-01  1.804265e-01  1.803962e-01  1.802949e-01 
##         68277        624701        623094        666528        319189 
##  1.800632e-01  1.799426e-01  1.799285e-01  1.799276e-01  1.797440e-01 
##     100043805         67008     100416335     100306944     102633002 
##  1.790653e-01  1.790545e-01  1.790440e-01  1.789023e-01  1.787849e-01 
##     100041006        666031         21393        667845         79263 
##  1.787164e-01  1.786617e-01  1.786332e-01  1.786091e-01  1.785599e-01 
##         67096        223770        672572     108167565        101943 
##  1.784870e-01  1.784430e-01  1.784202e-01  1.782812e-01  1.782416e-01 
##        668624         70385        330502        627833     100217457 
##  1.781927e-01  1.778526e-01  1.778136e-01  1.778042e-01  1.776218e-01 
##        328516         16533     100040970        328734         75406 
##  1.775912e-01  1.775590e-01  1.775557e-01  1.775518e-01  1.770405e-01 
##        240063        667170         15469        272411        231386 
##  1.770029e-01  1.769874e-01  1.769763e-01  1.767440e-01  1.762304e-01 
##         21808        641340     115489634        628119         57258 
##  1.761721e-01  1.761295e-01  1.760699e-01  1.760302e-01  1.759237e-01 
##     100504308         67046        622832     102637787         17898 
##  1.758134e-01  1.757318e-01  1.753578e-01  1.753431e-01  1.752881e-01 
##        628327     100048895         15114        213389         75480 
##  1.747355e-01  1.746234e-01  1.744486e-01  1.743430e-01  1.742308e-01 
##         26425        545867         22694     100038353         72454 
##  1.741234e-01  1.740989e-01  1.737644e-01  1.731997e-01  1.730204e-01 
##         72640        269424     100217462        666146        238130 
##  1.728378e-01  1.728290e-01  1.728236e-01  1.728174e-01  1.728086e-01 
##        327954        654823     102634273        433862     100042173 
##  1.724452e-01  1.723142e-01  1.721943e-01  1.721392e-01  1.720600e-01 
##         97268     100504088         77056        381823        666571 
##  1.720430e-01  1.718569e-01  1.715126e-01  1.713139e-01  1.709158e-01 
##         75482        319334     100042450     115486339         71846 
##  1.707122e-01  1.707052e-01  1.700210e-01  1.695206e-01  1.692175e-01 
##     100043797     100037396        218506        116905        269695 
##  1.690821e-01  1.689078e-01  1.686767e-01  1.685005e-01  1.678692e-01 
##         14700         14081     100418228     100504538     115489953 
##  1.677153e-01  1.676105e-01  1.674391e-01  1.672976e-01  1.672124e-01 
##     102466847         68364         68118     100418044        619653 
##  1.669825e-01  1.669516e-01  1.666653e-01  1.666217e-01  1.665743e-01 
##     115489016         67767     100044526         74570     102636600 
##  1.663754e-01  1.659590e-01  1.658356e-01  1.657435e-01  1.656063e-01 
##         77034         21807     100126821         73046     102632857 
##  1.653519e-01  1.653401e-01  1.652933e-01  1.651707e-01  1.649959e-01 
##         13390         72351        665412         51788        242408 
##  1.649943e-01  1.649123e-01  1.647680e-01  1.646875e-01  1.644599e-01 
##         24086         20708         75007     100039163        666677 
##  1.644228e-01  1.643777e-01  1.643478e-01  1.643162e-01  1.639984e-01 
##        329369     100328574         22763         75990     100042763 
##  1.639575e-01  1.639548e-01  1.638110e-01  1.635523e-01  1.624721e-01 
##     100043084        666300     100040795         17160        666009 
##  1.624143e-01  1.623375e-01  1.621784e-01  1.621336e-01  1.620218e-01 
##        231125         66242     100039992     100048941         77922 
##  1.619168e-01  1.618676e-01  1.618649e-01  1.618409e-01  1.615691e-01 
##         19893         68852     102640811        665548        233168 
##  1.614700e-01  1.613085e-01  1.609050e-01  1.608001e-01  1.603854e-01 
##        319822        629499        234684         73068     100041748 
##  1.601965e-01  1.598716e-01  1.596260e-01  1.593508e-01  1.592608e-01 
##        320184        380863         60504     100416327        319172 
##  1.591753e-01  1.591363e-01  1.587778e-01  1.586563e-01  1.586082e-01 
##     102640588         72083     100417723        668591         66197 
##  1.584812e-01  1.577288e-01  1.575387e-01  1.574222e-01  1.571000e-01 
##        228359         66671         20681        233887         67038 
##  1.570547e-01  1.569518e-01  1.566734e-01  1.566284e-01  1.564738e-01 
##         74044         74334         73183        723902     100416701 
##  1.563828e-01  1.560593e-01  1.560306e-01  1.560147e-01  1.559940e-01 
##        668363        211945         14058         13929         75354 
##  1.557157e-01  1.556688e-01  1.556187e-01  1.555832e-01  1.554769e-01 
##        622082         14430         72244         67797        432946 
##  1.553161e-01  1.551694e-01  1.551015e-01  1.549237e-01  1.546676e-01 
##        666488        384461         69692         68337         78369 
##  1.544988e-01  1.543589e-01  1.537431e-01  1.536486e-01  1.535808e-01 
##         53334         56632         75973        243853        668630 
##  1.534647e-01  1.533150e-01  1.532772e-01  1.529890e-01  1.529783e-01 
##        331188        629359        258783         28126        238091 
##  1.527257e-01  1.525227e-01  1.524681e-01  1.524078e-01  1.521942e-01 
##         67618     100417606         19771        434437         67725 
##  1.521503e-01  1.518200e-01  1.517968e-01  1.516943e-01  1.516646e-01 
##     100504056         12662     102635341        104010        233870 
##  1.511705e-01  1.510916e-01  1.510898e-01  1.510484e-01  1.503973e-01 
##        627792         76062         93705         69902        268390 
##  1.502861e-01  1.502679e-01  1.502188e-01  1.500378e-01  1.495459e-01 
##         69804     100417499     102633973        224619        320095 
##  1.495259e-01  1.494617e-01  1.494070e-01  1.492679e-01  1.492297e-01 
##         68273         66377        625672        110012        665773 
##  1.490934e-01  1.486988e-01  1.486039e-01  1.485417e-01  1.484580e-01 
##        623554     100039037        329307         69036        433813 
##  1.481300e-01  1.480450e-01  1.475764e-01  1.473668e-01  1.473126e-01 
##        667636     100503575        320171        627235     100302648 
##  1.472929e-01  1.470542e-01  1.469701e-01  1.465730e-01  1.465110e-01 
##         20276        232807        227094         54484        238024 
##  1.456195e-01  1.452671e-01  1.451262e-01  1.450777e-01  1.449648e-01 
##        140477         24050        230279        665497        545332 
##  1.447210e-01  1.445525e-01  1.442951e-01  1.442754e-01  1.441198e-01 
##         27756     100504267        622962        107392         67057 
##  1.440716e-01  1.438724e-01  1.438361e-01  1.433956e-01  1.433821e-01 
##        213550         67433        243833         14061         21818 
##  1.433277e-01  1.428532e-01  1.428174e-01  1.424644e-01  1.422459e-01 
##     115488997         69337     100040052        433458     105245929 
##  1.416181e-01  1.416113e-01  1.415995e-01  1.415051e-01  1.409718e-01 
##     100416045        665709         17131         70314        245944 
##  1.408739e-01  1.405918e-01  1.402929e-01  1.400428e-01  1.397254e-01 
##         12519        383769        445261        240895         11722 
##  1.397133e-01  1.395075e-01  1.394613e-01  1.394249e-01  1.388922e-01 
##     108168274     108168827     105243011        619597         19268 
##  1.388012e-01  1.383543e-01  1.383293e-01  1.382835e-01  1.381838e-01 
##        653029     100294507        675514         72121         69871 
##  1.380668e-01  1.380014e-01  1.379458e-01  1.378147e-01  1.377916e-01 
##         83382         11419        665180         69048        670795 
##  1.373994e-01  1.372045e-01  1.370985e-01  1.370975e-01  1.370248e-01 
##         93871         22166         19228     100044633        110119 
##  1.370060e-01  1.365355e-01  1.363638e-01  1.363523e-01  1.360471e-01 
##         83434        319181     100417130         74407         22750 
##  1.359126e-01  1.358792e-01  1.358669e-01  1.356529e-01  1.352842e-01 
##         18209        320637        338523     100417922         72776 
##  1.350992e-01  1.348706e-01  1.345567e-01  1.344150e-01  1.341570e-01 
##        233893     100043115        231805     100416024     101055641 
##  1.339072e-01  1.338825e-01  1.336573e-01  1.335332e-01  1.335285e-01 
##        545339     102634300     100417821     108167793     102640772 
##  1.334198e-01  1.333724e-01  1.330638e-01  1.328590e-01  1.325053e-01 
##     100042211     115490273         56214     100416123         77925 
##  1.323203e-01  1.322749e-01  1.321407e-01  1.320086e-01  1.319206e-01 
##         20557        666909        107730         75727     100041720 
##  1.318453e-01  1.315128e-01  1.313220e-01  1.313038e-01  1.312697e-01 
##         72131         12452         13144        621119         18656 
##  1.310966e-01  1.307622e-01  1.307061e-01  1.303702e-01  1.303573e-01 
##         12013     101056035         73453     115490288     105243511 
##  1.303243e-01  1.301597e-01  1.300564e-01  1.299936e-01  1.297232e-01 
##        735277        232223     105246501     118027464        230451 
##  1.293947e-01  1.292827e-01  1.292183e-01  1.291763e-01  1.290628e-01 
##     100416793        234686         20877        627119         66720 
##  1.290584e-01  1.290241e-01  1.289103e-01  1.287998e-01  1.285295e-01 
##         14608        101185         75030     100038373         68614 
##  1.283741e-01  1.283415e-01  1.282974e-01  1.282258e-01  1.280562e-01 
##         76642        435640     100417490        665191         12040 
##  1.277958e-01  1.277540e-01  1.276196e-01  1.275439e-01  1.274959e-01 
##         70726        327987     105242682         17524     102638607 
##  1.274863e-01  1.274684e-01  1.273759e-01  1.273299e-01  1.271184e-01 
##         16147         14376        353172         68918        634939 
##  1.271118e-01  1.269921e-01  1.269285e-01  1.268965e-01  1.267457e-01 
##         27052         11908     100039695         76469         17252 
##  1.266838e-01  1.265333e-01  1.264557e-01  1.261470e-01  1.260477e-01 
##         66256        628477     100039837     108168101         78825 
##  1.258709e-01  1.258570e-01  1.257649e-01  1.255328e-01  1.251553e-01 
##         67300        667793         54646         69713     100504432 
##  1.251268e-01  1.251179e-01  1.249539e-01  1.249003e-01  1.247212e-01 
##         11950         66435         74325         13409        385820 
##  1.246923e-01  1.246730e-01  1.244667e-01  1.243528e-01  1.242201e-01 
##        224912     100039110         66257         21817     100040471 
##  1.241156e-01  1.238959e-01  1.235358e-01  1.230188e-01  1.229362e-01 
##     102633750         66399     115487111         18020     100042584 
##  1.228782e-01  1.228072e-01  1.224256e-01  1.221092e-01  1.219512e-01 
##     101055724     115488066     100041265        235606        636070 
##  1.218440e-01  1.217709e-01  1.217470e-01  1.216142e-01  1.210903e-01 
##        210808         28084        110175     100416138     100039532 
##  1.210713e-01  1.209499e-01  1.208570e-01  1.208375e-01  1.205614e-01 
##     100043826        665426        244237        102247     100417350 
##  1.203157e-01  1.201562e-01  1.200720e-01  1.200389e-01  1.198420e-01 
##        215951        665601         75793         68922        625174 
##  1.198375e-01  1.195645e-01  1.194789e-01  1.193929e-01  1.192765e-01 
##     108168030        385658     100504637         19935        670994 
##  1.189865e-01  1.189704e-01  1.187062e-01  1.186481e-01  1.185664e-01 
##         19285         12569         21877         70821         68236 
##  1.184767e-01  1.183541e-01  1.182106e-01  1.180648e-01  1.177640e-01 
##     105245780        627110     100417999         21374         12096 
##  1.175890e-01  1.171602e-01  1.171280e-01  1.170998e-01  1.169340e-01 
##        676984        631429         22318        665564         66847 
##  1.166154e-01  1.165980e-01  1.162089e-01  1.161941e-01  1.161408e-01 
##     100862258         21390     100043787         78808         21648 
##  1.159060e-01  1.158323e-01  1.157807e-01  1.157766e-01  1.156886e-01 
##        330830        414359        269878        626870        228765 
##  1.156006e-01  1.154884e-01  1.154528e-01  1.153194e-01  1.149981e-01 
##        230793         21848        240117        241074        791415 
##  1.148242e-01  1.148102e-01  1.147860e-01  1.147650e-01  1.146770e-01 
##        231252     100039681         13057         18542     100038624 
##  1.145014e-01  1.144987e-01  1.143365e-01  1.141815e-01  1.140310e-01 
##         15444        383571        668589         73748        112422 
##  1.135143e-01  1.135025e-01  1.132638e-01  1.132394e-01  1.131940e-01 
##        668034        109136        623409        546818        208618 
##  1.131780e-01  1.130760e-01  1.129091e-01  1.127473e-01  1.126831e-01 
##     102635447         14432     102635154        236010        669557 
##  1.126629e-01  1.124392e-01  1.120758e-01  1.120529e-01  1.118680e-01 
##         76508        665749        102193         52469        242603 
##  1.115446e-01  1.115276e-01  1.115250e-01  1.111386e-01  1.110950e-01 
##        237320         71991        103573         13870     115489679 
##  1.110292e-01  1.108782e-01  1.108654e-01  1.108591e-01  1.107795e-01 
##        667373         27643        654360        668192         77453 
##  1.107666e-01  1.106558e-01  1.105506e-01  1.103760e-01  1.101879e-01 
##     115485864        382137         70004         70448        433940 
##  1.101859e-01  1.101425e-01  1.097714e-01  1.097546e-01  1.095144e-01 
##        666444        268490        636680         22017        626661 
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##         56620         18103     100042148     108168111         78934 
##  1.084656e-01  1.079635e-01  1.074826e-01  1.074453e-01  1.073575e-01 
##         67525        623859         73430        620140         74238 
##  1.072204e-01  1.071857e-01  1.071420e-01  1.070470e-01  1.070301e-01 
##         18828     100040239     100417983        384643        666633 
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##     100043462        225155         72748        232566     100041621 
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##     100039720     100040525     102640578        235542         22294 
##  1.053210e-01  1.052731e-01  1.051873e-01  1.051314e-01  1.049003e-01 
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##  1.046971e-01  1.045705e-01  1.045331e-01  1.039695e-01  1.038844e-01 
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##  1.037929e-01  1.036044e-01  1.034693e-01  1.034282e-01  1.033229e-01 
##        623601         12889        791363        667005        414093 
##  1.032027e-01  1.030372e-01  1.030218e-01  1.027305e-01  1.026541e-01 
##        385326        223417         15161        228787        633093 
##  1.026283e-01  1.026149e-01  1.025485e-01  1.023978e-01  1.020936e-01 
##        667218        625347        217893         72026        319591 
##  1.019739e-01  1.018932e-01  1.015914e-01  1.013885e-01  1.012953e-01 
##         75717         93893         20438         75475     105246847 
##  1.012003e-01  1.011613e-01  1.009765e-01  1.008323e-01  1.007522e-01 
##        114479         66174        212439        654809     100504235 
##  1.005774e-01  1.004990e-01  1.002784e-01  1.001358e-01  1.000470e-01 
##     100040260     100039748         66208        434858         14275 
##  1.000307e-01  9.988651e-02  9.981404e-02  9.959999e-02  9.947554e-02 
##        668806     108168917         13660        667617        665726 
##  9.851537e-02  9.806783e-02  9.805492e-02  9.804572e-02  9.791130e-02 
##         54409     100039979         71776        104369         66202 
##  9.773961e-02  9.769175e-02  9.767146e-02  9.741942e-02  9.687762e-02 
##        780884        665823        664903        638502     102633588 
##  9.662439e-02  9.632938e-02  9.625561e-02  9.578514e-02  9.566913e-02 
##     115490072     100034662        634662        240068        101490 
##  9.559447e-02  9.516839e-02  9.501018e-02  9.500062e-02  9.490767e-02 
##        664980     100034726        223626        319734         54127 
##  9.479492e-02  9.475713e-02  9.453059e-02  9.447999e-02  9.442830e-02 
##        668063     100503584     100417939        114664        383818 
##  9.441750e-02  9.413227e-02  9.395424e-02  9.360756e-02  9.296050e-02 
##     100418291     100040893        269999         68512        666194 
##  9.292137e-02  9.267588e-02  9.264099e-02  9.263400e-02  9.251785e-02 
##     105244530        383341     102632213     100039787        625342 
##  9.197833e-02  9.193261e-02  9.191356e-02  9.190232e-02  9.172478e-02 
##        666470        224224     100417556        246196        110958 
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##         56642         14050        623346         74322         13017 
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##     100044193     100037283        791299        624710        406209 
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##     102466887         77867         71046         68528         69714 
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##         52592     102639145         11778     102632831        277692 
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##        338346         74931        666153         70238         19871 
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##  8.396581e-02  8.387359e-02  8.374978e-02  8.366995e-02  8.345143e-02 
##        258117         71523     115486497         56426         20111 
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##  8.153977e-02  8.147938e-02  8.132441e-02  8.103232e-02  8.095686e-02 
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##        433745        214764     105243696        434175     100417576 
##  7.690279e-02  7.683073e-02  7.650939e-02  7.634103e-02  7.592840e-02 
##     102632854        666267         19215        382743         11891 
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##     102637985        328825        619308         80289        327951 
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##  7.308601e-02  7.304107e-02  7.262644e-02  7.261855e-02  7.255993e-02 
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##     102466802     100043010         14120         80749     102639290 
##  7.157704e-02  7.132944e-02  7.127967e-02  7.092548e-02  7.090064e-02 
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##        625929         12821        277333        383665        383186 
##  7.003873e-02  6.978874e-02  6.968518e-02  6.959531e-02  6.944957e-02 
##         18569        666011         17909        434186        208292 
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##  6.774273e-02  6.751547e-02  6.746362e-02  6.735730e-02  6.732732e-02 
##        666015         21339        107770        225655         67640 
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##  6.656168e-02  6.641618e-02  6.628226e-02  6.626338e-02  6.618380e-02 
##     100040766     105246638         66910     100417585     100313512 
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##     100038389        214973     100217441        216821        621875 
##  6.534130e-02  6.522497e-02  6.516100e-02  6.512869e-02  6.494463e-02 
##         26900     115489055     115489645        243725         76432 
##  6.488665e-02  6.488596e-02  6.465350e-02  6.457828e-02  6.442778e-02 
##     108167767        622870     100503737     102632479         50771 
##  6.440638e-02  6.422069e-02  6.419131e-02  6.412364e-02  6.404651e-02 
##        665655        630857        668481     115488812        668289 
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##         16971        216185     105245970         12493         21422 
##  6.267354e-02  6.262110e-02  6.252423e-02  6.250360e-02  6.225321e-02 
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##        245828         16950         22276     100534376         66694 
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##  5.307067e-02  5.303766e-02  5.287744e-02  5.266763e-02  5.221151e-02 
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## -2.013423e+01 -2.026166e+01 -2.040641e+01 -2.040728e+01 -2.043180e+01 
##         11370         11532        328329         71795         16506 
## -2.054519e+01 -2.065655e+01 -2.066751e+01 -2.067884e+01 -2.080227e+01 
##        110197         21753        246177         68977         21402 
## -2.081885e+01 -2.082343e+01 -2.089660e+01 -2.099222e+01 -2.104045e+01 
##         29819         54354         67771         17688         19241 
## -2.108422e+01 -2.118178e+01 -2.123755e+01 -2.130314e+01 -2.131678e+01 
##         66912         64291         19889        110611        211922 
## -2.134413e+01 -2.145314e+01 -2.146131e+01 -2.149964e+01 -2.152514e+01 
##         11758         12366        330959         50932         19091 
## -2.158824e+01 -2.171028e+01 -2.183609e+01 -2.184980e+01 -2.187004e+01 
##         13136         13655         12380        242687         20308 
## -2.198389e+01 -2.201806e+01 -2.202091e+01 -2.204133e+01 -2.205948e+01 
##         67949        226652        230863         12847        109151 
## -2.208233e+01 -2.209670e+01 -2.211590e+01 -2.219280e+01 -2.226579e+01 
##         20444         18477         76843         56619         17347 
## -2.232633e+01 -2.242786e+01 -2.243003e+01 -2.250136e+01 -2.257649e+01 
##        634731         12499         57265         67102        320404 
## -2.259847e+01 -2.259915e+01 -2.262391e+01 -2.262639e+01 -2.272856e+01 
##        329154        434341         65079        232334         13211 
## -2.282490e+01 -2.284115e+01 -2.287024e+01 -2.296826e+01 -2.300086e+01 
##        226861        320982         59287         16765         98193 
## -2.300771e+01 -2.302881e+01 -2.315084e+01 -2.315643e+01 -2.315913e+01 
##         19192         21391        216080         66385         16592 
## -2.322330e+01 -2.322875e+01 -2.323937e+01 -2.332596e+01 -2.340527e+01 
##        216543         68839         11566        210293         18193 
## -2.351684e+01 -2.362566e+01 -2.367942e+01 -2.372100e+01 -2.375293e+01 
##         27371        381290         59090         12469         76123 
## -2.385463e+01 -2.386226e+01 -2.387822e+01 -2.395388e+01 -2.400113e+01 
##         52463         67707         66505         58246         68616 
## -2.412388e+01 -2.427703e+01 -2.433314e+01 -2.435580e+01 -2.438863e+01 
##         16565         75974         66151         74116        109032 
## -2.456665e+01 -2.458458e+01 -2.470273e+01 -2.471517e+01 -2.483064e+01 
##         14701        101994        328232        192176         77976 
## -2.494384e+01 -2.503396e+01 -2.522001e+01 -2.524921e+01 -2.531907e+01 
##        230761         21885         18637        320586        215690 
## -2.532055e+01 -2.542386e+01 -2.558271e+01 -2.578931e+01 -2.596623e+01 
##        207278        226562        232288        102098         67664 
## -2.596658e+01 -2.605682e+01 -2.606733e+01 -2.607622e+01 -2.633259e+01 
##         74081         12774        227333         16870         17159 
## -2.639080e+01 -2.644380e+01 -2.662501e+01 -2.662923e+01 -2.664267e+01 
##         20365         15441         13713         16192         19062 
## -2.666812e+01 -2.680573e+01 -2.683988e+01 -2.687570e+01 -2.690871e+01 
##         14026         74464         12925        195727         12798 
## -2.707306e+01 -2.709763e+01 -2.721152e+01 -2.724725e+01 -2.736126e+01 
##         73469        233405         16019        213539         20230 
## -2.745295e+01 -2.746790e+01 -2.751809e+01 -2.761785e+01 -2.761792e+01 
##        214597         66514        193385        269224         19073 
## -2.772229e+01 -2.780737e+01 -2.792900e+01 -2.798563e+01 -2.809123e+01 
##        236511         72042         64214         70160         18032 
## -2.828440e+01 -2.830905e+01 -2.836098e+01 -2.838189e+01 -2.857485e+01 
##         19253         15381         70186        170753         20661 
## -2.886372e+01 -2.897296e+01 -2.905180e+01 -2.909125e+01 -2.911469e+01 
##         76709        394432        245595        192119         13032 
## -2.912533e+01 -2.920517e+01 -2.933037e+01 -2.952980e+01 -2.955863e+01 
##         16541         17215         16579         18566         13714 
## -2.967891e+01 -2.970448e+01 -2.972509e+01 -2.973313e+01 -2.973655e+01 
##         68944         14964         68514         18106         19125 
## -2.981447e+01 -2.982794e+01 -2.984584e+01 -2.986352e+01 -3.010941e+01 
##         21346        268445         17434         24064        108829 
## -3.014037e+01 -3.022344e+01 -3.028257e+01 -3.028525e+01 -3.034041e+01 
##        328795         69035         11545        226841        381694 
## -3.034309e+01 -3.042580e+01 -3.056458e+01 -3.059438e+01 -3.065353e+01 
##         22355         20649         93840         14131         18203 
## -3.073458e+01 -3.089179e+01 -3.108135e+01 -3.108374e+01 -3.111849e+01 
##         22778         67460         15488         66824         73341 
## -3.119150e+01 -3.133982e+01 -3.158023e+01 -3.161124e+01 -3.164245e+01 
##         80898        382045         12479        229473         14645 
## -3.204381e+01 -3.212607e+01 -3.218028e+01 -3.221080e+01 -3.221508e+01 
##         22341         20363        140580         11774     100038725 
## -3.238451e+01 -3.267617e+01 -3.277156e+01 -3.291925e+01 -3.306979e+01 
##         74513        108147     102636203         67869         16398 
## -3.307213e+01 -3.308863e+01 -3.333354e+01 -3.334727e+01 -3.345675e+01 
##        216197         20658         52440         19663         78334 
## -3.361876e+01 -3.364743e+01 -3.374171e+01 -3.408345e+01 -3.413617e+01 
##        103841        226432         72465        140577     100039888 
## -3.420663e+01 -3.468042e+01 -3.518511e+01 -3.522517e+01 -3.537463e+01 
##        319670        230316         19651         74106         17993 
## -3.577501e+01 -3.581375e+01 -3.603205e+01 -3.647545e+01 -3.651021e+01 
##         76088     101056162        170737        259302         93966 
## -3.659563e+01 -3.691719e+01 -3.741105e+01 -3.749811e+01 -3.791699e+01 
##        330286         70945         77065         16576         52882 
## -3.807434e+01 -3.828043e+01 -3.913264e+01 -3.917426e+01 -3.926784e+01 
##        229905         55927         20849         14194        103677 
## -3.938657e+01 -4.017073e+01 -4.018758e+01 -4.026193e+01 -4.048411e+01 
##         22719        226830        269233         22083         14972 
## -4.053104e+01 -4.071213e+01 -4.090484e+01 -4.107216e+01 -4.124494e+01 
##         19395         71787         17219         66043        667799 
## -4.140105e+01 -4.179203e+01 -4.192427e+01 -4.201288e+01 -4.209862e+01 
##         74185         15002        545030         93736         59054 
## -4.217274e+01 -4.233988e+01 -4.239183e+01 -4.241247e+01 -4.244234e+01 
##         17260        108672         20588        215243         72462 
## -4.303193e+01 -4.308185e+01 -4.326826e+01 -4.341543e+01 -4.392390e+01 
##         22344         66892        219140         18019        108686 
## -4.418905e+01 -4.445410e+01 -4.452748e+01 -4.466339e+01 -4.477381e+01 
##         22350        218693         72433         17685         71330 
## -4.570632e+01 -4.593650e+01 -4.689054e+01 -4.740028e+01 -4.839698e+01 
##         13171        225207         27494         56229         67792 
## -4.913579e+01 -4.935576e+01 -4.978452e+01 -4.992187e+01 -5.003554e+01 
##         53623        217946         56878        668218         16451 
## -5.035323e+01 -5.074829e+01 -5.093589e+01 -5.155879e+01 -5.161069e+01 
##         20452         76041         19265         20742         66725 
## -5.227720e+01 -5.229611e+01 -5.258472e+01 -5.265596e+01 -5.267994e+01 
##         16432         12986         66307         70155         19649 
## -5.287157e+01 -5.322065e+01 -5.335488e+01 -5.393285e+01 -5.486249e+01 
##         56526         71592         76707         76429         14744 
## -5.524985e+01 -5.538930e+01 -5.553155e+01 -5.595173e+01 -5.601732e+01 
##         64213        233328        223697        226594         78798 
## -5.648619e+01 -5.708142e+01 -5.732178e+01 -5.743436e+01 -5.788853e+01 
##         14525         26914        268973        240752         73690 
## -5.812591e+01 -5.828190e+01 -5.884724e+01 -5.984129e+01 -6.057751e+01 
##         12557         12183         22596         83925         26432 
## -6.079037e+01 -6.139320e+01 -6.517781e+01 -6.631423e+01 -6.720011e+01 
##         70729         67392         83669         18201        108116 
## -6.744012e+01 -6.848802e+01 -6.963742e+01 -7.025400e+01 -7.044124e+01 
##         27412        107684        106522        170748         14025 
## -7.193323e+01 -7.319603e+01 -7.326184e+01 -7.494057e+01 -7.595600e+01 
##         56792        218629        171170         50766        236915 
## -7.777856e+01 -8.095908e+01 -8.134638e+01 -8.210289e+01 -8.324326e+01 
##         27357        108052         17304         12523         54204 
## -8.419715e+01 -8.645548e+01 -8.740168e+01 -8.938799e+01 -9.459737e+01 
##         13007        240754         70549         11732         50934 
## -9.546244e+01 -9.615622e+01 -9.667404e+01 -9.819803e+01 -1.030445e+02 
##         76438         17085        244202         56228         16985 
## -1.095454e+02 -1.109358e+02 -1.131828e+02 -1.149241e+02 -1.156694e+02 
##         12506         18583         13641         76132         12490 
## -1.176728e+02 -1.240534e+02 -1.290506e+02 -1.330333e+02 -1.332771e+02 
##         72198        252838         11600        665574        330450 
## -1.358195e+02 -1.377297e+02 -1.392665e+02 -1.573439e+02 -1.641227e+02 
##         75547        107895        104099         53328        216848 
## -1.695769e+02 -1.707981e+02 -2.127129e+02 -3.076527e+02 -3.076527e+02

You can also do GSEA with String. Strings needs a 2 column Table, first column with IDs, second with value that rank genes. We can use the ranked vector named with Entrez IDs generated before. We write it out, and then upload the file to String. Select “Proteins with Values/Ranks” in the Search Menu, and upload the table. Confirm mapping of IDs.
You will get the sig. enriched Terms from different databases. You can see the rank of the genes in the enriched terms in the ranked list by selecting the specific term.

5 homework

5.1 perform analysis with STAT3-wt vs. dsRed; compare results to results obtained already in this skript with Y640F vs. dsRed

  • perform these ORAs for STAT3-wt vs. dsRed:
    • for Reactome (=enrichPathway function)
    • for MsigDB Hallmark (=enricher function)
  • perform GSEA analysis for STAT3-wt vs. dsRed:
    • for KEGG (=gseKEGG function)
    • for MsigDB Hallmark (=GSEA function)
  • perform SPIA analysis for STAT3-wt vs. dsRed:

From ORAs and GSEA select results (Y640F.vs.dsRed and STAT3.vs.dsRed) from one database (the same, or different - ideally the one with a appropriate number of pathways, or the one where you can best interpret the results).

In presentation, put ORAs from different Databases of STAT3-wt.vs.dsRed and Y640F.vs.dsRed next to each other, and shortly describe what you see (dont mind if your interpretation is correct or not - it is sometimes hard to say, without additional exploration of results).

Alternatively, you can use compareCluster to directly compare both analysis in one plot.

5.2 perform ORAs, GSEA, SPIA with genes up-reg. by y640f but not/down-reg. by stat3-wt

  • just use one of the 3 methods, to see what we get from this analysis, as compared with indirect comparisons done above